Immunoglobulins and uses thereof

ABSTRACT

The present invention relates to a monoclonal antibody platform designed to be coupled to therapeutic peptides to increase the half-life of the therapeutic peptide in a subject. The invention also relates to pharmaceutical compositions and methods for use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/413,613, filed on Oct. 27, 2016, and U.S. Provisional PatentApplication No. 62/413,586 filed on Oct. 27, 2016. Each disclosure isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed generally to a novel antibody orfragments thereof, and uses thereof as carriers to be coupled to atherapeutic peptide to increase the half-life of the therapeutic peptidein vivo. The invention also relates to pharmaceutical compositions andmethods for use thereof.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “PRD3459 Sequence Listing” and a creation date of Oct. 23,2017, and having a size of 20 kb. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety. In the event of any inconsistency with regardto the structures for SEQ ID NOs: 1-27 between the information describedherein and the Sequence Listing submitted electronically via EFS-Webwith a file name “PRD3459 Sequence Listing,” the information herein willprevail.

BACKGROUND OF THE INVENTION

Peptide-based therapeutics are often explored and developed as they canprovide the specificity and selectivity, as evidenced by the manyapproved peptide-based treatments, but the ways in which they can beused are limited by their relatively short in vivo half-lives. Therehave been numerous half-life extension strategies evaluated and utilizedwith peptide-based therapeutics. Antibodies or antibody fragments havebeen used as half-life extension moieties for pharmacologically activemoieties to prevent or mitigate rapid in vivo elimination of thepharmacologically active moieties.

There is a need for an improved immunoglobulin that can be used as apharmacologically inactive half-life extension moiety forpharmacologically active moieties, preferably the peptide-basedtherapeutics.

The foregoing discussion is presented solely to provide a betterunderstanding of the nature of the problems confronting the art andshould not be construed in any way as an admission as to prior art norshould the citation of any reference herein be construed as an admissionthat such reference constitutes “prior art” to the instant application.

BRIEF SUMMARY OF THE INVENTION

In one general aspect, the present invention relates to an isolatedmonoclonal antibody or antigen-binding fragment thereof comprising aheavy chain complementarity determining region 1 (HCDR1), a HCDR2, aHCDR3, and a light chain complementarity determining region 1 (LCDR1), aLCDR2, and a LCDR3, having the polypeptide sequences of SEQ ID NO: 16,17, 18, 19, 20, and 21, respectively.

In a preferred embodiment, an isolated monoclonal antibody orantigen-binding fragment thereof of the invention comprises a heavychain variable domain (VH) having the polypeptide sequence of SEQ IDNO:12, and a light chain variable domain (VL) having the polypeptidesequence of SEQ ID NO:14. More preferably, an isolated monoclonalantibody of the invention comprises a heavy chain (HC) having thepolypeptide sequence of SEQ ID NO:13, and a light chain (LC) having thepolypeptide sequence of SEQ ID NO:15.

The invention also relates to an isolated nucleic acid encoding amonoclonal antibody or antigen-binding fragment thereof of theinvention; a vector, preferably an expression vector, comprising thenucleic acid; and a host cell comprising the vector. Also provided is amethod of producing an isolated monoclonal antibody or antigen bindingfragment thereof of the invention, the method comprising culturing ahost cell of the invention under conditions to produce the monoclonalantibody or antigen binding fragment thereof, and recovering theantibody or antigen-binding fragment thereof from the cell or culture.

Embodiments of the invention include a monoclonal antibody orantigen-binding fragment thereof of the invention conjugated to at leastone pharmacologically active moiety, preferably a therapeutic peptide,directly or via a linker. The monoclonal antibody or antigen-bindingfragment thereof of the invention can be conjugated to any therapeuticpeptide. Examples of the therapeutic peptide include, but are notlimited to, oxyntomodulin, glucagon-like peptide 1 (GLP1), peptidetyrosine tyrosine (PYY), exendin (exenatide), amylin (pramlintide),alpha-melanocyte stimulating hormone (MSH), cocaine- andamphetamine-regulated transcript (CART), neuropeptide Y receptor Y1(NPY1) antagnoists, neuropeptide Y receptor Y5 (NPY5) antagonists,neurotensin S, neuropeptide B, neuropeptide W, ghrelin, bombesin-likereceptor 3 (BRS3), galanin, cholecystokinin (CCK), orexin,melanin-concentrating hormone (MCH), oxytocin, and stresscopin.

Also provided is a method of producing a monoclonal antibody orantigen-binding fragment thereof of the invention conjugated to atherapeutic peptide, comprising reacting an electrophile, preferablybromoacetamide or maleimide, introduced onto a sidechain of thetherapeutic peptide, with a sulfhydryl group, preferably the sulfhydrylgroup of the cysteine residue of SEQ ID NO:18, of the monoclonalantibody or antigen-binding fragment thereof, thereby creating acovalent linkage between the therapeutic peptide and the monoclonalantibody or antigen-binding fragment thereof.

The invention also relates to a pharmaceutical composition comprising amonoclonal antibody or antigen-binding fragment thereof of the inventionconjugated to at least one pharmacologically active moiety, preferably atherapeutic peptide, directly or via a linker, and a pharmaceuticallyacceptable carrier.

Another general aspect of the invention relates to a method ofincreasing the half-life of a therapeutic peptide in a subject, themethod comprising conjugating the therapeutic peptide with a monoclonalantibody or antigen-binding fragment thereof comprising a heavy chaincomplementarity determining region 1 (HCDR1), a HCDR2, a HCDR3, and alight chain complementarity determining region 1 (LCDR1), a LCDR2, and aLCDR3, having the polypeptide sequences of SEQ ID NO: 16, 17, 18, 19,20, and 21, respectively, wherein the therapeutic peptide is conjugatedto the monoclonal antibody or antigen-binding fragment thereof at asulfhydryl group, preferably the sulfhydryl group of the Cys residue ofSEQ ID NO:18.

Further aspects, features and advantages of the present invention willbe better appreciated upon a reading of the following detaileddescription of the invention and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofpreferred embodiments of the present application, will be betterunderstood when read in conjunction with the appended drawings. Itshould be understood, however, that the application is not limited tothe precise embodiments shown in the drawings.

FIG. 1: Shows a general peptide-mAb conjugation strategy according to anembodiment of the invention. X represents an electrophile introducedonto a sidechain of a therapeutic peptide, such as bromoacetamide ormaleimide, that reacts site specifically with the sulfhydryl group ofthe Cys residue engineered into a CDR of the half-life extending mAb,creating a covalent linkage between the therapeutic peptide and the mAb.

FIG. 2: Summary of CDR residues selected for substitution in PH9H5_VH(SEQ ID NO:4) and in PH9L3_VL (SEQ ID NO:3). Residues substituted withCys are bolded and underlined.

FIG. 3: Shows the structure of compound 1 (SEQ ID NO:2).

FIG. 4: Pharmacokinetics of the compound 1 in DIO mice.

FIG. 5: Pharmacokinetics of the compound 1 in cynomolgus monkeys.

FIG. 6: Food intake in DIO mice treated with the compound 1: acutedosing.

FIG. 7: Weight loss in DIO mice treated with the compound 1: acutedosing.

FIG. 8: Food intake in DIO mice treated with the compound 1: chronicdosing.

FIG. 9: Weight loss in DIO mice treated with the compound 1: chronicdosing.

FIG. 10: Shows a reaction scheme to produce a monoclonal antibodyoxyntomodulin (mAb-OXM) compound according to an embodiment of theinvention. The top reaction is a reduction of the disulfide at Cys102 ofthe mAb (e.g., MSCB97). The bottom reaction is a conjugation of thereduced mAb with the OXM peptide variant. R is cysteine or glutathione.Cys102 on the heavy chain of MSCB97 is shown with flanking amino acidsas their single letter codes. OXM refers to the amino acid portion ofthe peptide variant. Aib2, Lys30 and the bromoacetylated discretepolyethyleneglycol (dPEG)12 spacer are shown. His1 of the mAb is shownas the single letter code.

FIG. 11: Shows the structure of compound 2 (SEQ ID NO:27).

FIG. 12: Shows a graph demonstrating the functional stability ofcompound 2 in human plasma over 168 hours. Percent remaining normalizedto time zero (t=0) of OXM peptide analog mAb conjugate compound 2▴,control 1, which had previously been demonstrated to be stable in exvivo human plasma ♦, control 2, which had previously been demonstratedto be unstable in ex vivo human plasma ● and ◯, and additional OXMpeptide analog mAb conjugates compound 3 (mAb conjugated withH-Aib-QGTFTSDYSKYLDERRARDFVEWLLNK-(COCH₂CH₂(OCH₂CH₂)₁₂NHCOCH₂Br)—NH₂(SEQ ID NO: 25))

and compound 4 (mAb conjugated withH-Aib-QGTFTSDYSKYLDERRARDFVEWLLNTK-(COCH₂CH₂(OCH₂CH₂)₁₂ NHCOCH₂Br)—NH₂(SEQ ID NO: 26))▾, over time in hours (hr) on the X-axis.

FIG. 13: Shows a graph demonstrating the functional stability ofcompound 2 in monkey plasma over 168 hours. Percent remaining normalizedto time zero (t=0) of compound 2 ▴, control 1 previously demonstrated tobe stable in ex vivo human plasma ♦, control 2 previously demonstratedto be unstable in ex vivo human plasma ● and ◯, and additional OXMpeptide analog mAb conjugates compound 3

and compound 4 ▾, over time in hours (hr) on the X-axis.

FIG. 14: Shows a graph demonstrating % change in food intake incynomolgus monkeys treated with compound 2.

FIG. 15: Shows a graph demonstrating % change in body weight incynomolgus monkeys treated with compound 2.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentrationor a concentration range described herein, are to be understood as beingmodified in all instances by the term “about.” Thus, a numerical valuetypically includes ±10% of the recited value. For example, aconcentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, aconcentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).As used herein, the use of a numerical range expressly includes allpossible subranges, all individual numerical values within that range,including integers within such ranges and fractions of the values unlessthe context clearly indicates otherwise.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the invention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers and are intended to be non-exclusive or open-ended.For example, a composition, a mixture, a process, a method, an article,or an apparatus that comprises a list of elements is not necessarilylimited to only those elements but can include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

It should also be understood that the terms “about,” “approximately,”“generally,” “substantially” and like terms, used herein when referringto a dimension or characteristic of a component of the preferredinvention, indicate that the described dimension/characteristic is not astrict boundary or parameter and does not exclude minor variationstherefrom that are functionally the same or similar, as would beunderstood by one having ordinary skill in the art. At a minimum, suchreferences that include a numerical parameter would include variationsthat, using mathematical and industrial principles accepted in the art(e.g., rounding, measurement or other systematic errors, manufacturingtolerances, etc.), would not vary the least significant digit.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences (e.g., cyclic PYY₃₋₃₆polypeptide sequences, oxyntomodulin polypeptide sequences, antibodylight chain or heavy chain sequences), refer to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms or by visual inspection using methodsknown in the art in view of the present disclosure.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

As used herein, “subject” means any animal, preferably a mammal, mostpreferably a human. The term “mammal” as used herein, encompasses anymammal. Examples of mammals include, but are not limited to, cows,horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs,monkeys, humans, etc., more preferably a human.

The term “administering” with respect to the methods of the invention,means a method for therapeutically or prophylactically preventing,treating or ameliorating a syndrome, disorder or disease as describedherein by using a conjugate of the invention or a form, composition ormedicament thereof. Such methods include administering an effectiveamount of said conjugate, conjugate form, composition or medicament atdifferent times during the course of a therapy or concurrently in acombination form. The methods of the invention are to be understood asembracing all known therapeutic treatment regimens.

The term “effective amount” means that amount of active conjugate orpharmaceutical agent that elicits the biological or medicinal responsein a tissue system, animal or human, that is being sought by aresearcher, veterinarian, medical doctor, or other clinician, whichincludes preventing, treating or ameliorating a syndrome, disorder, ordisease being treated, or the symptoms of a syndrome, disorder ordisease being treated.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts.

As used herein the term “coupled” refers to the joining or connection oftwo or more objects together. When referring to chemical or biologicalcompounds, coupled can refer to a covalent connection between the two ormore chemical or biological compounds. By way of a non-limiting example,an antibody of the invention can be coupled with a peptide of interestto form an antibody coupled peptide. An antibody coupled peptide can beformed through specific chemical reactions designed to conjugate theantibody to the peptide. In certain embodiments, an antibody of theinvention can be covalently coupled with a peptide of the inventionthrough a linker. The linker can, for example, be first covalentlyconnected to the antibody or the peptide, then covalently connected tothe peptide or the antibody.

As used herein, the term “linker” refers to a chemical module comprisinga covalent or atomic chain that covalently attaches a peptide to anantibody. The linker can, for example, include, but is not limited to, apeptide linker, a hydrocarbon linker, a polyethylene glycol (PEG)linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, apolyester linker, a hybrid linker consisting of PEG and an embeddedheterocycle, and a hydrocarbon chain. The PEG linkers can, for example,comprise 2-24 PEG units.

As used herein, the term “conjugate” refers to an antibody or a fragmentthereof covalently coupled to a pharmaceutically active moiety. The term“conjugated to” refers to an antibody or a fragment thereof of inventioncovalently linked to or covalently connected to a pharmaceuticallyactive moiety, preferably a therapeutic peptide, directly or indirectlyvia a linker. By way of a non-limiting example, the antibody can be amonoclonal antibody of the invention and the pharmaceutically activemoiety can be a therapeutic peptide, such as a cyclic PYY, anoxyntomodulin peptide, or any other therapeutic peptide of interest. Thepharmaceutically active moiety can also be a non-peptide organic moiety(i.e., “small molecule”). In the present disclosure, with respect to anantibody or antigen binding fragment thereof according to an embodimentof the invention, the phrase “a conjugate comprising an antibody orantigen binding fragment thereof and a pharmaceutically active moiety(or therapeutic peptide) conjugated thereto” is used interchangeablywith the phrase “an antibody or antigen binding fragment thereofconjugated to a pharmaceutically active moiety (or a therapeuticpeptide).”

The peptide sequences described herein are written according to theusual convention whereby the N-terminal region of the peptide is on theleft and the C-terminal region is on the right. Although isomeric formsof the amino acids are known, it is the L-form of the amino acid that isrepresented unless otherwise expressly indicated.

Antibodies

In one general aspect, the invention relates to a novel antibody, whichhas been engineered to be non-targeting and to contain a cysteineresidue capable of being used to chemically conjugate (i.e., couple) apharmaceutically active moiety, such as a therapeutic peptide (e.g., acyclic PYY peptide, an oxyntomodulin or variant peptide, etc.), in asite-specific manner, such that the antibody coupled peptide has anextended/increased half-life compared to the peptide alone. As usedherein, the term “non-targeting” in the context of an antibody refers toan antibody that does not specifically bind to any target in vivo. Asused herein, an antibody that “specifically binds to a target” refers toan antibody that binds to a target antigen, with a KD of 1×10⁻⁷ M orless, preferably 1×10⁻⁸M or less, more preferably 5×10⁻⁹ M or less,1×10⁻⁹M or less, 5×10⁻¹⁰ M or less, or 1×10⁻¹⁰ M or less. The term “KD”refers to the dissociation constant, which is obtained from the ratio ofKd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M). KDvalues for antibodies can be determined using methods in the art in viewof the present disclosure. For example, the KD of an antibody can bedetermined by using surface plasmon resonance, such as by using abiosensor system, e.g., a Biacore® system, or by using bio-layerinterferometry technology, such as a Octet RED96 system. The smaller thevalue of the KD of an antibody, the higher affinity that the antibodybinds to a target antigen.

Monoclonal antibodies, complete or a fragment thereof, can be used as ahalf-life extending moiety. Monoclonal antibodies are well-studiedproteins that have been utilized and characterized for uses in vivo, andas such, the mechanisms that enable their protracted half-life in vivoand the mechanisms for their elimination in vivo are well understood.Additionally, the spatial separation and presentation of the two “arms”of the monoclonal antibody can be advantageous for effective bivalentpresentation of a therapeutic moiety (i.e., a therapeutic peptide).Therapeutics in which toxins or other small molecule drugs arechemically linked to a monoclonal antibody have been developed buttypically utilize a monoclonal antibody that binds to a specific antigenand targets the antibody-drug conjugate to a tissue/cell of interest,which preferentially expressed the antigen, and typically the drug/smallmolecule is attached to the antibody in a manner that does not impactantigen binding of the antibody.

For therapeutic peptide-mAb conjugates, antigen specific binding by thehalf-life extending monoclonal antibody is not desired. Because of this,a heavy chain (HC) and light chain (LC) variable (V) domain pair notexpected to specifically bind any target are used for preparing thecoupling-enabled, non-targeting monoclonal antibody of the invention. Toobtain a coupling-enabled, non-targeting monoclonal antibody, a cysteineresidue is engineered into one of the complementarity determiningregions (CDRs) of a selected non-targeting antibody. Thepharmaceutically active moiety (e.g., therapeutic peptide/compound) cancontain the appropriate chemical moiety to allow for the conjugation ofthe pharmaceutically active moiety to the engineered cysteine residue ofthe non-targeting monoclonal antibody. A peptide-monoclonal antibodygeneral conjugation strategy according to an embodiment of the inventionis shown in FIG. 1.

The term “antibodies” as used herein is meant in a broad sense andincludes non-human (e.g., murine, rat), human, human-adapted, humanizedand chimeric monoclonal antibodies, antibody fragments, bispecific ormultispecific antibodies, dimeric, tetrameric or multimeric antibodies,and single chain antibodies.

Antibody light chains of any vertebrate species can be assigned to oneof two clearly distinct types, namely kappa (κ) and lambda (λ), based onthe amino acid sequences of their constant domains. Accordingly, theantibodies of the invention can contain a kappa or lambda light chainconstant domain. According to particular embodiments, the antibodies ofthe invention include heavy and/or light chain constant regions, e.g.,from mouse or human antibodies. In addition to the heavy and lightconstant domains, antibodies contain an antigen-binding region that ismade up of a light chain variable region and a heavy chain variableregion, each of which contains three domains (i.e., complementaritydetermining regions 1-3; (CDR1, CDR2, and CDR3)). The light chainvariable region domains are alternatively referred to as LCDR1, LCDR2,and LCRD3, and the heavy chain variable region domains are alternativelyreferred to as HCDR1, HCDR2, and HCDR3.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD,IgE, IgG and IgM, depending on the heavy chain constant domain aminoacid sequence. IgG is the most stable of the five types ofimmunoglobulins, having a serum half-life in humans of about 23 days.IgA and IgG are further sub-classified as the isotypes IgA₁, IgA₂, IgG₁,IgG₂, IgG₃ and IgG₄. Each of the four IgG subclasses has differentbiological functions known as effector functions. These effectorfunctions are generally mediated through interaction with the Fcreceptor (FcγR) or by binding C1q and fixing complement. Binding to FcγRcan lead to antibody dependent cell mediated cytolysis, whereas bindingto complement factors can lead to complement mediated cell lysis. Anantibody of the invention utilized for its ability to extend half-lifeof a therapeutic peptide has no or minimal effector function, butretains its ability to bind FcRn, the binding of which can be a primarymeans by which antibodies have an extended in vivo half-life.

In one embodiment, the invention relates to an isolated antibody orantigen binding fragment thereof comprising a light chain variableregion having completely human Ig germline V gene sequences, and a heavychain variable region having completely human Ig germline V genesequences except HCDR3 having the amino acid sequence of SEQ ID NO:18,wherein the antibody or antigen binding fragment thereof does notspecifically bind to any human antigen in vivo. In certain embodiments,the invention relates to an isolated antibody or antigen bindingfragment thereof comprising a light chain variable region havingcompletely human Ig germline V gene sequences, and a heavy chainvariable region having completely human Ig germline V gene sequencesexcept HCDR3 having the amino acid sequence of SEQ ID NO:18, wherein theantibody or antigen binding fragment thereof does not specifically bindto any human antigen in vivo, wherein the isolated antibody or antigenbinding fragment thereof is coupled to a pharmaceutically active moiety(e.g., a cyclic PYY peptide, an oxyntomodulin peptide, and/or atherapeutic peptide of the invention).

As used herein, the term “antigen-binding fragment” refers to anantibody fragment such as, for example, a diabody, a Fab, a Fab′, aF(ab′)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a(dsFv)₂, a bispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody(ds diabody), a single-chain antibody molecule (scFv), a single domainantibody (sdab) an scFv dimer (bivalent diabody), a multispecificantibody formed from a portion of an antibody comprising one or moreCDRs, a camelized single domain antibody, a nanobody, a domain antibody,a bivalent domain antibody, or any other antibody fragment that binds toan antigen but does not comprise a complete antibody structure. Anantigen-binding fragment is capable of binding to the same antigen towhich the parent antibody or a parent antibody fragment binds. Accordingto particular embodiments, the antigen-binding fragment comprises alight chain variable region, a light chain constant region, and an Fdsegment (i.e., portion of the heavy chain which is included in the Fabfragment). According to other particular embodiments, theantigen-binding fragment comprises Fab and F(ab′).

As used herein, the term “single-chain antibody” refers to aconventional single-chain antibody in the field, which comprises a heavychain variable region and a light chain variable region connected by ashort peptide of about 15 to about 20 amino acids. As used herein, theterm “single domain antibody” refers to a conventional single domainantibody in the field, which comprises a heavy chain variable region anda heavy chain constant region or which comprises only a heavy chainvariable region.

The phrase “isolated antibody or antibody fragment” refers to anantibody or antibody fragment that is substantially free of otherantibodies having different antigenic specificities (e.g., an isolatedantibody specifically binding a target antigen is substantially free ofantibodies that specifically do not bind the target antigen). Moreover,an isolated antibody or antibody fragment can be substantially free ofother cellular material and/or chemicals.

An antibody variable region consists of a “framework” region interruptedby three “antigen binding sites”. The antigen binding sites are definedusing various terms: (i) Complementarity Determining Regions (CDRs),three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1,LCDR2, LCDR3), are based on sequence variability (Wu and Kabat J Exp Med132:211-50, 1970; Kabat et al Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991). (ii) “Hypervariable regions,” “HVR,” or “HV,”three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3), refer tothe regions of an antibody variable domains which are hypervariable instructure as defined by Chothia and Lesk (Chothia and Lesk Mol Biol196:901-17, 1987). Other terms include “IMGT-CDRs” (Lefranc et al., DevComparat Immunol 27:55-77, 2003) and “Specificity Determining ResidueUsage” (SDRU) (Almagro Mol Recognit 17:132-43, 2004). The InternationalImMunoGeneTics (IMGT) database (http://www_mgt_org) provides astandardized numbering and definition of antigen-binding sites. Thecorrespondence between CDRs, HVs and IMGT delineations is described inLefranc et al., Dev Comparat Immunol 27:55-77, 2003.

“Framework” or “framework sequences” are the remaining sequences of avariable region other than those defined to be antigen binding sites.Because the antigen binding sites can be defined by various terms asdescribed above, the exact amino acid sequence of a framework depends onhow the antigen-binding site was defined.

In one embodiment of the invention, an isolated antibody or antigenbinding fragment thereof comprises a light chain variable region havingthe LCDR1, LCDR2 and LCDR3 of the amino acid sequence of SEQ ID NO: 19,SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and a heavy chainvariable region having the HCDR1, HCDR2 and HCDR3 of the amino acidsequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18,respectively.

In another embodiment, the isolated antibody further comprises a Fcregion derived from human IgG4 Fc region. Human IgG4 Fc region hasreduced ability to bind FcγR and complement factors compared to otherIgG sub-types. Preferably, the Fc region contains human IgG4 Fc regionhaving substitutions that eliminate effector function. Thus, theisolated antibody further comprises a Fc region having a modified humanIgG4 Fc region containing one or more of the following substitutions:substitution of proline for glutamate at residue 233, alanine or valinefor phenylalanine at residue 234 and alanine or glutamate for leucine atresidue 235 (EU numbering, Kabat, E. A. et al. (1991) Sequences ofProteins of Immunological Interest, 5th Ed. U.S. Dept. of Health andHuman Services, Bethesda, Md., NIH Publication no. 91-3242). Removingthe N-linked glycosylation site in the IgG4 Fc region by substitutingAla for Asn at residue 297 (EU numbering) is another way to ensure thatresidual effector activity is eliminated.

Preferably, an antibody of the invention exists as dimers joinedtogether by disulfide bonds and various non-covalent interactions. Thus,the Fc portion useful for the antibody of the invention can be humanIgG4 Fc region containing a substitution, such as serine to proline atposition at 228 (EU numbering), that stabilizes heavy chain dimerformation and prevents the formation of half-IgG4 Fc chains.

In another embodiment, the C-terminal Lys residue in the heavy chain isremoved, as commonly seen in recombinantly produced monoclonalantibodies.

“Human antibody” refers to an antibody having heavy and light chainvariable regions in which both the framework and the antigen bindingsites are derived from sequences of human origin. If the antibodycontains a constant region, the constant region also is derived fromsequences of human origin.

Human antibody comprises heavy or light chain variable regions that are“derived from” sequences of human origin if the variable regions of theantibody are obtained from a system that uses human germlineimmunoglobulin or rearranged immunoglobulin genes. Such systems includehuman immunoglobulin gene libraries displayed on phage, and transgenicnon-human animals such as mice carrying human immunoglobulin loci asdescribed herein. “Human antibody” may contain amino acid differenceswhen compared to the human germline or rearranged immunoglobulinsequences due to for example naturally occurring somatic mutations orintentional introduction of substitutions in the framework or antigenbinding sites. Typically, “human antibody” is at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an aminoacid sequence encoded by a human germline or rearranged immunoglobulingene. In some cases, “human antibody” may contain consensus frameworksequences derived from human framework sequence analyses, for example asdescribed in Knappik et al., J Mol Biol 296:57-86, 2000), or syntheticHCDR3 incorporated into human immunoglobulin gene libraries displayed onphage, for example as described in Shi et al., J Mol Biol 397:385-96,2010 and Intl. Pat. Publ. No. WO2009/085462). Antibodies in whichantigen binding sites are derived from a non-human species are notincluded in the definition of “human antibody”.

Isolated antibodies according to embodiment of the invention can besynthetic. The antibodies, while derived from human immunoglobulinsequences, can be generated using systems such as phage displayincorporating synthetic CDRs and/or synthetic frameworks, or can besubjected to in vitro mutagenesis to improve antibody properties,resulting in antibodies that do not naturally exist within the humanantibody germline repertoire in vivo.

The term “recombinant antibody” as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal for human immunoglobulin genes or ahybridoma prepared therefrom, antibodies isolated from a host celltransformed to express the antibody, antibodies isolated from arecombinant, combinatorial antibody library, and antibodies prepared,expressed, created or isolated by any other means that involve splicingof human immunoglobulin gene sequences to other DNA sequences, orantibodies that are generated in vitro using Fab arm exchange.

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules of a single molecular composition. A monoclonalantibody composition displays a single binding specificity and affinityfor a particular epitope, or in a case of a bispecific monoclonalantibody, a dual binding specificity to two distinct epitopes. Themonoclonal antibodies of the invention can be made by the hybridomamethod, phage display technology, single lymphocyte gene cloningtechnology, or by recombinant DNA methods. For example, the monoclonalantibodies can be produced by a hybridoma which includes a B cellobtained from a transgenic nonhuman animal, such as a transgenic mouseor rat, having a genome comprising a human heavy chain transgene and alight chain transgene.

In certain embodiments, the term “mAb” refers to a monoclonal antibody.In one embodiment, the mAb has a heavy chain variable region (VH)sequence comprising SEQ ID NO:12 and a light chain variable region (VL)sequence comprising SEQ ID NO:14. In certain embodiments the mAb is afully human monoclonal antibody having a heavy chain (HC) sequencecomprising SEQ ID NO:13 and a light chain (LC) sequence comprising SEQID NO:15. In certain embodiments, the lysine residue at position 446 ofSEQ ID NO:13 is optionally missing.

As used herein, the term “chimeric antibody” refers to an antibodywherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. The variable region of both the lightand heavy chains often corresponds to the variable region of an antibodyderived from one species of mammal (e.g., mouse, rat, rabbit, etc.)having the desired specificity, affinity, and capability, while theconstant regions correspond to the sequences of an antibody derived fromanother species of mammal (e.g., human) to avoid eliciting an immuneresponse in that species.

As used herein, the term “multispecific antibody” refers to an antibodythat comprises a plurality of immunoglobulin variable domain sequences,wherein a first immunoglobulin variable domain sequence of the pluralityhas binding specificity for a first epitope or comprises germlinesequences lacking any known binding specificity and a secondimmunoglobulin variable domain sequence of the plurality has bindingspecificity for a second epitope or comprises germline sequences lackingany known binding specificity, and wherein the first and/or secondimmunoglobulin variable domain optionally include a conjugatedpharmaceutically active moiety (e.g., a therapeutic peptide). In anembodiment, the first and second epitopes are on the same antigen, e.g.,the same protein (or subunit of a multimeric protein). In an embodiment,the first and second epitopes overlap or substantially overlap. In anembodiment, the first and second epitopes do not overlap or do notsubstantially overlap. In an embodiment, the first and second epitopesare on different antigens, e.g., the different proteins (or differentsubunits of a multimeric protein). In an embodiment, the first andsecond immunoglobulin variable domains include the same conjugatedpharmaceutically active moiety. In an embodiment, the first and secondimmunoglobulin variable domains include different pharmaceuticallyactive moieties. In an embodiment, only the first immunoglobulinvariable domain includes a conjugated pharmaceutically active moiety. Inan embodiment, only the second immunoglobulin variable domain includes aconjugated pharmaceutically active moiety. In an embodiment, amultispecific antibody comprises a third, fourth, or fifthimmunoglobulin variable domain. In an embodiment, a multispecificantibody is a bispecific antibody molecule, a trispecific antibody, or atetraspecific antibody molecule.

As used herein, the term “bispecifc antibody” refers to a multispecificantibody that binds no more than two epitopes or two antigens and/orcomprises two conjugated pharmaceutically active moieties (e.g., thesame or different pharmaceutically active moiety). A bispecific antibodyis characterized by a first immunoglobulin variable domain sequencewhich has binding specificity for a first epitope or comprises germlinesequences lacking any known binding specificity and a secondimmunoglobulin variable domain sequence that has binding specificity fora second epitope or comprises germline sequences lacking any knownbinding specificity, and wherein the first and/or second immunoglobulinvariable domain optionally include a conjugated pharmaceutically activemoiety. In an embodiment, the first and second epitopes are on the sameantigen, e.g., the same protein (or subunit of a multimeric protein). Inan embodiment, the first and second epitopes overlap or substantiallyoverlap. In an embodiment the first and second epitopes are on differentantigens, e.g., the different proteins (or different subunits of amultimeric protein). In an embodiment, the first and secondimmunoglobulin variable domains include the same conjugatedpharmaceutically active moiety. In an embodiment, the first and secondimmunoglobulin variable domains include different pharmaceuticallyactive moieties. In an embodiment, only the first immunoglobulinvariable domains includes a conjugated pharmaceutically active moiety.In an embodiment, only the second immunoglobulin variable domainincludes a conjugated pharmaceutically active moiety. In an embodiment abispecific antibody comprises a first heavy chain variable domainsequence and light chain variable domain sequence which have bindingspecificity for a first epitope or comprise germline sequences lackingany known binding specificity and a second heavy chain variable domainsequence and light chain variable domain sequence which have bindingspecificity for a second epitope or comprise germline sequences lackingany known binding specificity, and wherein the first and/or second heavychain variable domains optionally include a conjugated pharmaceuticallyactive moiety. In an embodiment, the first and second heavy chainvariable domains include the same conjugated pharmaceutically activemoiety. In an embodiment, the first and second heavy chain variabledomains include different conjugated pharmaceutically active moieties.In an embodiment, only the first heavy chain variable domain includes aconjugated pharmaceutically active moiety. In an embodiment, only thesecond heavy chain variable domain includes a conjugatedpharmaceutically active moiety.

“Full length antibody” as used herein refers to an antibody having twofull length antibody heavy chains and two full length antibody lightchains. A full length antibody heavy chain (HC) consists of a heavychain variable region (VH) and constant domains (CH1, CH2, and CH3). Afull length antibody light chain (LC) consists of a light chain variableregion (VL) and constant domain (CL). The full length antibody can belacking the C-terminal lysine (K) in either one or both heavy chains.

The term “Fab-arm” or “half molecule” refers to one heavy chain-lightchain pair.

Full length bispecific antibodies can be generated for example using Fabarm exchange (or half molecule exchange) between two monospecificbivalent antibodies by introducing substitutions at the heavy chain CH3interface in each half molecule to favor heterodimer formation of twoantibody half molecules having distinct specificity either in vitro incell-free environment or using co-expression. The Fab arm exchangereaction is the result of a disulfide-bond isomerization reaction anddissociation-association of CH3 domains. The heavy-chain disulfide bondsin the hinge regions of the parent monospecific antibodies are reduced.The resulting free cysteines of one of the parent monospecificantibodies form an inter heavy-chain disulfide bond with cysteineresidues of a second parent monospecific antibody molecule andsimultaneously CH3 domains of the parent antibodies release and reformby dissociation-association. The CH3 domains of the Fab arms may beengineered to favor heterodimerization over homodimerization. Theresulting product is a bispecific antibody having two Fab arms or halfmolecules which each can bind a distinct epitope.

“Homodimerization” as used herein, with respect to the antibodies,refers to an interaction of two heavy chains having identical CH3 aminoacid sequences. “Homodimer” as used herein, with respect to theantibodies, refers to an antibody having two heavy chains with identicalCH3 amino acid sequences.

“Heterodimerization” as used herein, with respect to the antibodies,refers to an interaction of two heavy chains having non-identical CH3amino acid sequences. “Heterodimer” as used herein, with respect to theantibodies, refers to an antibody having two heavy chains withnon-identical CH3 amino acid sequences.

The “knob-in-hole” strategy (see, e.g., PCT Intl. Publ. No. WO2006/028936) can be used to generate full length bispecific antibodies.Briefly, selected amino acids forming the interface of the CH3 domainsin human IgG can be mutated at positions affecting CH3 domaininteractions to promote heterodimer formation. An amino acid with asmall side chain (hole) is introduced into a heavy chain of an antibodyspecifically binding a first antigen and an amino acid with a large sidechain (knob) is introduced into a heavy chain of an antibodyspecifically binding a second antigen. After co-expression of the twoantibodies, a heterodimer is formed as a result of the preferentialinteraction of the heavy chain with a “hole” with the heavy chain with a“knob”. Exemplary CH3 substitution pairs forming a knob and a hole are(expressed as modified position in the first CH3 domain of the firstheavy chain/modified position in the second CH3 domain of the secondheavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T,T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

Other strategies such as promoting heavy chain heterodimerization usingelectrostatic interactions by substituting positively charged residuesat one CH3 surface and negatively charged residues at a second CH3surface may be used, as described in US Pat. Publ. No. US2010/0015133;US Pat. Publ. No. US2009/0182127; US Pat. Publ. No. US2010/028637 or USPat. Publ. No. US2011/0123532. In other strategies, heterodimerizationmay be promoted by following substitutions (expressed as modifiedposition in the first CH3 domain of the first heavy chain/modifiedposition in the second CH3 domain of the second heavy chain):L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V,T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, orT350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in U.S.Pat. Publ. No. US2012/0149876 or U.S. Pat. Publ. No. US2013/0195849.

In addition to methods described above, bispecific antibodies can begenerated in vitro in a cell-free environment by introducingasymmetrical mutations in the CH3 regions of two monospecifichomodimeric antibodies and forming the bispecific heterodimeric antibodyfrom two parent monospecific homodimeric antibodies in reducingconditions to allow disulfide bond isomerization according to methodsdescribed in Intl. Pat. Publ. No. WO2011/131746. In the methods, thefirst monospecific bivalent antibody and the second monospecificbivalent antibody are engineered to have certain substitutions at theCH3 domain that promoter heterodimer stability; the antibodies areincubated together under reducing conditions sufficient to allow thecysteines in the hinge region to undergo disulfide bond isomerization;thereby generating the bispecific antibody by Fab arm exchange. Theincubation conditions may optimally be restored to non-reducing.Exemplary reducing agents that may be used are 2-mercaptoethylamine(2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione,tris(2-carboxyethyl)phosphine (TCEP), L-cysteine andbeta-mercaptoethanol, preferably a reducing agent selected from thegroup consisting of: 2-mercaptoethylamine, dithiothreitol andtris(2-carboxyethyl)phosphine. For example, incubation for at least 90min at a temperature of at least 20° C. in the presence of at least 25mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH offrom 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

The numbering of amino acid residues in the antibody constant regionthroughout the specification is performed according to the EU index asdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991), unless otherwise explicitly stated.

Conjugates

In another general aspect, the invention relates to a conjugatecomprising an antibody of the invention covalently conjugated to apharmaceutically active moiety, such as a synthetic therapeutic peptide(e.g., a cyclic PYY peptide or an oxyntomodulin variant peptide), in asite-specific manner, such that the antibody coupled peptide has anextended/increased half-life compared to the peptide alone. Theconjugates are useful for preventing, treating, or ameliorating diseasesor disorders disclosed herein. The invention also relates topharmaceutical compositions, methods of preparation and methods for usethereof.

In certain embodiments, the antibody of the invention is modified tocomprise at least one cysteine residue substitution that is capable ofbeing conjugated to the pharmaceutically active moiety toextend/increase the half-life of the pharmaceutically active moiety. Incertain embodiments, the at least one cysteine residue substitution iscomprised in a complementarity determining region of the antibody. Incertain embodiments, the at least one cysteine residue substitution isin a heavy chain complementarity determining region (HCDR). In certainembodiments, the at least one cysteine residue substitution is in anHCDR3, wherein the HCDR3 comprises an amino acid sequence of SEQ IDNO:18. In certain embodiments, the antibody comprising an HCDR3comprising an amino acid sequence of SEQ ID NO:18 has at least oneadditional cysteine substitution that is capable of being conjugated tothe pharmaceutically active moiety.

In certain embodiments the pharmaceutically active moiety can comprise alinker. The linker can be modified chemically to allow for theconjugation of the antibody to the pharmaceutically active moiety. Thelinker can, for example, include, but is not limited to, a peptidelinker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, apolypropylene glycol (PPG) linker, a polysaccharide linker, a polyesterlinker, a hybrid linker consisting of PEG and an embedded heterocycle,and a hydrocarbon chain. The PEG linkers can, for example, comprise 2-24PEG units.

In certain embodiments, a monoclonal antibody of the invention isconjugated to one, two, three, four, five, or six pharmaceuticallyactive moieties (e.g., therapeutic peptide(s)) of interest. In preferredembodiments, the non-targeting monoclonal antibody is conjugated to twopharmaceutically active moieties of interest. In certain embodimentswhere the monoclonal antibody is conjugated to at least twopharmaceutically active moieties of interest, the pharmaceuticallyactive moieties of interest can be the same pharmaceutically activemoiety or can be different pharmaceutically active moieties.

Methods of conjugating antibodies of the invention with thepharmaceutically active moieties of the invention are known in the art.Briefly, the antibodies of the invention can be reduced with a reducingagent (e.g., TCEP (tris(2-carboxyethyl) phosphine), purified (e.g., byprotein A adsorption or gel filtration), and conjugated with thepharmaceutically active moiety (e.g., by providing a lyophilized peptideto the reduced antibody under conditions that allow for conjugation).After the conjugation reaction, the conjugate can be purified by ionexchange chromatography or hydrophobic interaction chromatography (HIC)with a final purification step of protein A adsorption. In certainembodiments, the antibodies of the invention can be purified prior tobeing reduced utilizing HIC methods. For more detailed description ofthe conjugation methods, see, e.g., Examples 3 and 7, and Dennler etal., Antibodies 4:197-224 (2015).

“Homodimerization” as used herein, with respect to the conjugates,refers to an interaction of two identical pharmaceutically activemoieties with the antibody. “Homodimer” as used herein, with respect tothe conjugate, refers to an antibody coupled to two identicalpharmaceutically active moieties.

“Heterodimerization” as used herein, with respect to the conjugates,refers to an interaction of two different pharmaceutically activemoieties with the antibody. “Heterodimer” as used herein, with respectto the conjugate, refers to an antibody coupled to two differentpharmaceutically active moieties.

Cyclic PYY Peptides

PYY₃₋₃₆ is an endogenous hormone secreted by L cells in the distal gutthat acts as an agonist of the Y2 receptor to inhibit food intake. Givenits role in controlling appetite and food intake as well as itsanti-secretory and pro-absorptive effects in the gastrointestinal tractin mammals, PYY₃₋₃₆ can be effective in treating obesity and associatedconditions as well as in a number of gastrointestinal disorders.However, the therapeutic utility of PYY₃₋₃₆ itself as a treatment agentis limited by its rapid metabolism and short circulating half-life.Thus, an antibody of the invention can be used as a carrier for aPYY₃₋₃₆, preferably a modified PYY₃₋₃₆, which extends the half-life ofthe PYY₃₋₃₆ peptide and reduces the metabolism of the peptide in vivo.

In certain embodiments of the invention, the modified PYY₃₋₃₆ peptidesare cyclic PYY peptides. The terms “cyclic PYY peptide,” “cyclic PYY₃₋₃₆analog,” and “cyclic PYY₃₋₃₆ peptide analog” can be usedinterchangeably. Examples of cyclic PYY peptides that can be used in theconjugates are described in U.S. Provisional Patent Application No.62/413,613, filed on Oct. 27, 2016, and U.S. patent application No.entitled “Cyclic peptide tyrosine tyrosine compounds as modulators ofneuropeptide receptors,” filed on the same day as this application withthe attorney docket number PRD3411, the contents of both applicationsare hereby incorporated by reference in their entireties.

Examples of conjugates comprising an antibody of the invention and acyclic PYY peptide are described in U.S. Provisional Patent ApplicationNo. 62/413,586, filed on Oct. 27, 2016, and U.S. patent application Ser.No. ______ entitled “Antibody coupled cyclic peptide tyrosine tyrosinecompounds as modulators of neuropeptide Y receptors” filed on the sameday as this application with the attorney docket number PRD3436, thecontents of both applications are hereby incorporated by reference intheir entireties. For example, in the cyclic PYY peptides, theN-terminal amino acid residue of the cycle links by way of its α-aminofunctionality to the linking group, which in turn connects to the sidechain residue of the amino acid at position 31 of the NTSC-PYY peptide.Lysine residues can be incorporated at various positions of the hPYY₃₋₃₆sequence to provide a convenient functional handle for furtherderivatization. The lysine residues can be modified to be coupled to themonoclonal antibody either directly or indirectly. In an indirectcoupling to the monoclonal antibody, the lysine residue can be modifiedto comprise a linker which will allow for the cyclic PYY peptide to becoupled to the monoclonal antibody. One skilled in the art willrecognize that related orthologues could also be effectively employed assuch and are contemplated herein.

Oxyntomodulin Peptides

Oxyntomodulin (OXM) is a 37 amino acid peptide secreted fromenteroendocrine L cells in the gut. Through its agonist activity atGLP-1 receptor (GLP1R) and glucagon receptor (GCGR), OXM enhances β-cellfunction, reduces food intake and enhances energy expenditure. Throughthese complementary mechanisms, OXM-mediated weight loss may besuperior, relative to currently marketed GLP1R agonists. OXM can alsoreduce plasma cholesterol and triglycerides. The half-life of OXM inhumans is very short, on the order of minutes (Schjoldager et al., Eur.J. Clin. Invest. 18(5):499-503 (1988)). Thus, an embodiment of theinvention relates to a conjugate comprising an antibody of the inventioncovalently linked to oxyntomodulin to provide dual agonist properties ofoxyntomodulin and a protracted half-life sufficient to achieveonce-weekly dosing.

Extending the peptide half-life is accomplished by stabilizing the OXMpeptide against susceptibility to proteolysis and by reducing plasmaclearance. For example, proteolysis by DPP4 was mitigated bysubstituting the serine at position 2 with aminoisobutyric acid (Aib).The helical topology of the peptide was stabilized by introducing abifurcated salt bridge from Q20R to S16E and Q24E, and a potentialoxidation liability was mitigated by substituting methionine 27 withleucine. The circulating lifetime of the peptide was increased bycovalent attachment of a monoclonal antibody (mAb). Such antibody-drugconjugates can exhibit prolonged plasma half-lives by virtue of theirsize, which can reduce glomerular filtration, and by recycling via theneonatal Fc receptor. A short oligoethylene glycol spacer was interposedbetween the mAb and the peptide to ensure unhindered access of thepeptide to GCGR and GLP1R.

According to embodiments of the invention, an oxyntomodulin conjugate ofthe present invention contains four characteristics. (A) Dual agonism:the oxyntomodulin conjugates have dual agonism at GLP-1 and glucagonreceptors. (B) Receptor balance: the oxyntomodulin conjugates do nothave excessive potency bias to either GLP1R or GCGR, as excessive biasto GLP1R could result in a conjugate in which GLP-1 mediatedgastrointestinal adverse events occur at escalating exposures prior toGCGR target engagement, and excessive bias to GCGR could mitigateglycemic efficacy. (C) Biodistribution: the oxyntomodulin conjugateshave a modest bias toward GLP-1 receptor potency (relative tooxyntomodulin alone), with the objective of achieving target engagementof peripheral GCGR and central, energy intake-modulating, GLP1R atsimilar exposures. (D) Once-weekly dosing: the oxyntomodulin conjugatesare competitive by being capable of being administered to a subject inneed thereof once weekly.

Other Therapeutic Peptides

Also provided herein are conjugates comprising other peptides that arecapable of being conjugated to the monoclonal antibody platformdescribed herein. The peptides can, for example, be selected from thegroup consisting of glucagon-like peptide 1 (GLP1), exendin (exenatide),amylin (pramlintide), alpha-melanocyte stimulating hormone (MSH),cocaine- and amphetamine-regulated transcript (CART), neuropeptide Yreceptor Y1 (NPY1) antagonists, neuropeptide Y receptor Y5 (NPY5)antagonists, neurotensin S, neuropeptide B, neuropeptide W, ghrelin,bombesin-like receptor 3 (BRS3), galanin, cholecystokinin (CCK), orexin,melanin-concentrating hormone (MCH), oxytocin, and stresscopin.

Glucagon-like peptide-1 (GLP-1) is a 30 amino acid long peptide hormonederived from the tissue-specific post-translational processing of theproglucagon gene. GLP-1 is produced and secreted by intestinalenteroendocrine L-cells and certain neurons upon food consumption. Theinitial product GLP-1 (1-37) is susceptible to amidation and proteolyticcleavage, which gives rise to two truncated and equipotent biologicallyactive forms, GLP-1 (7-36) amide and GLP-1 (7-37). Endogenous GLP-1 israpidly degraded through multiple routes, primarily by dipeptidylpeptidase-4 (DPP-4 or DPP-IV), but also neutral endopeptidase 24.11 (NEP24.11) and through renal clearance, which results in a half-life ofapproximately 2 minutes. GLP-1 based treatments have been associatedwith weight loss and lower hypoglycemia risks, which are important inthe treatment of type II diabetes.

Exendin (exenatide) is a GLP-1 agonist belonging to a group of incretinmimetics, which have been approved for the treatment of type II diabetesmellitus (T2DM). Exenatide is a synthetic version of exendin-4, a39-amino acid peptide hormone, which is an insulin secretagogue withglucoregulatory effects. Exendin-4 share extensive homology and functionwith mammalian GLP-1, but has a therapeutic advantage as it is resitantto degradation by DPP-4 (DPP-IV), which allows for a longerpharmacological half-life. The biological characteristics of exendin-4led to the consideration for use as a treatment in T2DM.

Amylin (islet amyloid polypeptide (IAPP)) is a 37 amino acid peptidehormone, which is cosecreted with insulin from the pancreatic β-cells.Amylin plays a role in glycemic regulation by slowing gastric emptyingand promoting satiety. Amylin (IAPP) is processed from an 89 amino acidpeptide. Proiset amyloid polypeptide (proIAPP), is produced in thepancreatic β-cells from the 89 amino acid peptide as a 67 amino acidpeptide after a 22 amino acid signal peptide is cleaved. It is believedthat impaired processing of proIAPP can lead to the conditions thatresult in type II diabetes, as the lack of production of amylin (IAPP)can lead to a lack of glycemic control. Pramlintide is an amylinomimeticagent and is at least as potent as human amylin. It is a 37-amino-acidpolypeptide and differs in amino acid sequence from human amylin byreplacement of amino acids with proline at positions 25 (alanine), 28(serine), and 29 (serine). The prolines are naturally occurringvariations found in rat amylin. As a result of these substitutions,pramlintide is soluble, nonadhesive, and nonaggregating, therebyovercoming a number of the physicochemical liabilities of native humanamylin (Janes et al., Diabetes 45(Suppl 2):235A (1996); Young et al.,Drug Dev. Res. 37:231-48 (1996b)).

α-Melanocyte-stimulating hormone (α-MSH) is an endogenous peptidehormone and neuropeptide of the melanocortin family. α-MSH is the mostimportant of the melanocyte-stimulating hormones (MSHs) in stimulatingmelanogenesis, which is a process in mammals responsible forpigmentation of the hair and skin. α-MSH also plays a role in feedingbehavior, energy homeostasis, sexual activity, and protection againstischemia and reperfusion injury.

Cocaine- and amphetamine-regulated transcript (CART) is a neuropeptideprotein encoded by the CARTPT gene in humans. CART peptides, inparticular CART (55-102), seems to have an important function in theregulation of energy homeostasis, as the CART peptides interact withseveral hypothalamic appetite circuits. CART expression is regulated byperipheral peptide hormones involved in appetite regulation, whichincludes leptin, cholecystokinin, and ghrelin. CART and cholecystokininhave synergistic effects on appetite regulation. It is believed thatCART peptides play a role in anxiety-like behavior, induced by ethanolwithdrawal; modulate the locomotor, conditioned place preference andcocaine self-administration effect of psychostimulants; inhibit foodintake; and are involved in fear and startile behavior. CARThypoactivity in the hypothalamus is associated with hyperphagia andweight gain, and CART is thought to play a role in the opioid mesolimbicdopamine circuit that modulates natural reward processes.

Neuropeptide Y (NPY) has numerous roles in the body that include, e.g.,the control of feeding behaviour, cortical neural activity, heartactivity and emotional regulation. NPY has also been implicated inseveral human diseases including obesity, alcoholism and depression.Furthermore, blockade of the central actions of NPY using anti-NPYantibodies, antisense oligodeoxynucleotides against NPY and NPY receptorantagonists results in a decrease in food intake in energy-deprivedanimals. In particular, Neuropeptide Y receptor Y5 (NPY5) andNeuropeptide Y receptor Y1 (NPY1) have been shown to stimulate differentphases of feeding (Br J Pharmacol. 2003 August; 139(8):1433-40). Thus,NPY5 and NPY1 antagonists could be effective in the treatment obesityand other related metabolic diseases.

Neurotensin is a 13 amino acid neuropeptide that is implicated in theregulation of luteinizing hormone and prolactin release and hassignificant interaction with the dopaminergic system. Neurotensin isdistributed throughout the central nervous system with the highestlevels being in the hypothalamus, amygdala, and nucleus accumbens.Neurotensin can induce a variety of effects, including analgesia,hypothermia, increased locomoter activity, and is involved in theregulation of dopamine pathways.

Neuropeptide B (NPB) is a short, biologically active peptide, whoseprecursor is encoded by the NBP gene. NPB can act via two Gprotein-coupled receptors, called neuropeptide B/W receptors 1 and 2(NPBWR1 and NPBWR2). It is believed that neuropeptide B is associatedwith the regulation of feeding, neuroendocrine system, memory, learning,and the afferent pain pathway.

Neuropeptide W (NPW) exists in two forms, consisting of 23 (NPW23) or 30(NPW30) amino acids. These neuropeptides bind to and can act via the twoG-protein couple receptors, NPBWR1 (a.k.a GPR7) and NPBWR2 (a.k.a.GPR8). NPW has been shown to suppress food intake and body weight and toincrease both heat production and body temperature, suggesting that NPWfunctions as an endogenous catabolic signaling molecule.

Ghrelin (a.k.a. ienomorelin (INN)) is a peptide hormone, produced byghrelinergic cells in the gastrointestinal tract, which functions as aneuropeptide in the central nervous system. Ghrelin plays a role inregulating appetite, regulating the distribution and rate of use ofenergy, regulating the reward perception in dopamine neurons. Ghrelin isencoded by the GHRL gene and is thought to be produced from the cleavageof the preproghrelin, which is cleaved to produce proghrelin, which isleaved to produce the 28 amino acid ghrelin. Unlike other endogenouspeptides, ghrelin is able to cross the blood-brain barrier, which givesexogenously administered ghrelin a unique clinical potential.

Bombesin-like receptor 3 (BRS3) is a G protein-couple receptor that onlyinteracts with known naturally occurring bombesin-related peptides withlow affinity, and, as it has no high-affinity ligand, BRS3 is classifiedas an orphan receptor.

Galanin is a neuropeptide encoding by the GAL gene. Galanin is widelyexpressed in the brain, spinal cord, and gut of humans as well as otheranimals. The function of galanin has yet to be fully classified;however, galanin is predominantly involved in the modulation andinhibition of action potentials in neurons. Galanin has been implicatedin many biologically diverse functions including, but not limited to,nociception, waking and sleep regulation, cognition, feeding, regulationof mood, and regulation of blood pressure. Galanin is often co-localizedwith classical neurotransmitters such as acetylcholine, serotonin, andnorepinephrine, and also with neuromodulators such as neuropeptide Y,substance P, and vasoactive intestinal peptide.

Cholecystokinin (CCK) is a peptide hormone of the gastrointestinalsystem responsible for stimulating the digestion of fat and protein. CCKis synthesized and secreted by the enteroendocrine cells in the smallintestine and its presence causes the release of digestive enzymes andbile from the pancreas and gallbladder. CCK plays a role in digestion,satiety, and anxiety.

Orexin (a.k.a. hypocretin) is a neuropeptide that regulates arousal,wakefulness, and appetite. There are two types of orexin: orexin A and B(hypocretin-1 and -2), hich are 33 and 28 amino acids in length,respectively. The orexin system was primarily believed to be involvedwith the stimulation of food intake, and in addition to the rolesdescribed above, orexins regulate energy expenditure ad modulatevisceral function.

Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid orexigenichypothalamic peptide, which is believed to be involved in the regulationof feeding behavior, mood, sleep-wake cycle, and energy balance. MCHexpressing neurons are located within the lateral hypothalamus and zonaincreta, and despite this restricted distribution, MCH neurons projectwidely throughout the brain.

Oxytocin is a peptide hormone and neuropeptide normally produced by theparaventricular nucleus of the hypothalamus and released by theposterior pituitary. Oxytocin is believed to play a role in socialbonding, sexual reproduction, and during and after childbirth. Theoxytocin receptor is a G protein-coupled receptor that requiresmagnesium and cholesterol and belongs to the rhodopsin-type (class I)group of G protein-coupled receptors.

Human stresscopin (h-SCP) is a 40-amino-acid peptide, that is a memberof the corticotrophin releasing hormone (CRH) peptide family. Thebiological actions of the CRH peptide family are elicited by two 7transmembrane G-protein coupled receptors, CRH receptor type 1 (CRHR1)and CRH receptor type 2 (CRHR2). Although these receptors contain highsequence homology, the different members of the CRH peptide familyexpress significant differences in their relative binding affinity,degree of receptor activation and selectivity for these two receptors.Unlike many of the CRH family members, h-SCP expresses greaterselectivity for the CRHR2 and acts as a mediator that aids in theprocess of attenuating the initiation and maintenance of physiologicalstress. In addition to its apparent role in physiological stress, h-SCPhas been reported to elicit a number of other physiological actions. Itexerts effects on the endocrine, central nervous, cardiovascular,pulmonary, gastrointestinal, renal, skeletal muscle, and inflammatorysystems. CRHR2 activity has also been implicated in skeletal musclewasting disease, such as sarcopenia, motor activity and food intake,participates in a cardioprotective role and expresses bronchorelaxantand anti-inflammatory activity. Furthermore, stresscopin mimics havebeen identified that are useful for treating medical indicationsmediated by corticotrophin releasing hormone receptor 2 activity see,e.g., U.S. Patent App. No.: 20100130424.

Half-Life Extending Moieties

In addition to the antibody of the present invention or an antigenbinding fragment thereof, the conjugates of the invention canincorporate one or more other moieties for extending the half-life ofthe pharmaceutical active moiety, for example via covalent interaction.Exemplary other half-life extending moieties include, but not limitedto, albumin, albumin variants, albumin-binding proteins and/or domains,transferrin and fragments and analogues thereof. Additional half-lifeextending moieties that can be incorporated into the conjugates of theinvention include, for example, polyethylene glycol (PEG) molecules,such as PEG5000 or PEG20,000, fatty acids and fatty acid esters ofdifferent chain lengths, for example laurate, myristate, stearate,arachidate, behenate, oleate, arachidonate, octanedioic acid,tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and thelike, polylysine, octane, carbohydrates (dextran, cellulose, oligo- orpolysaccharides) for desired properties. These moieties can be directfusions with the protein scaffold coding sequences and can be generatedby standard cloning and expression techniques. Alternatively, well knownchemical coupling methods can be used to attach the moieties torecombinantly and chemically produced conjugates of the invention.

A pegyl moiety can, for example, be added to the peptide molecules ofthe invention by incorporating a cysteine residue to the C-terminus ofthe molecule and attaching a pegyl group to the cysteine using wellknown methods.

Peptide molecules of the invention incorporating additional moieties canbe compared for functionality by several well-known assays. For example,the biological or pharmacokinetic activities of a therapeutic peptide ofinterest, alone or in a conjugate according to the invention, can beassayed using known in vitro or in vivo assays and compared.

Pharmaceutical Compositions

In another general aspect, the invention relates to a pharmaceuticalcomposition, comprising the conjugates of the invention and apharmaceutically acceptable carrier. The term “pharmaceuticalcomposition” as used herein means a product comprising a conjugate ofthe invention together with a pharmaceutically acceptable carrier.Conjugates of the invention and compositions comprising them are alsouseful in the manufacture of a medicament for therapeutic applicationsmentioned herein.

As used herein, the term “carrier” refers to any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipidcontaining vesicle, microsphere, liposomal encapsulation, or othermaterial well known in the art for use in pharmaceutical formulations.It will be understood that the characteristics of the carrier, excipientor diluent will depend on the route of administration for a particularapplication. As used herein, the term “pharmaceutically acceptablecarrier” refers to a non-toxic material that does not interfere with theeffectiveness of a composition according to the invention or thebiological activity of a composition according to the invention.According to particular embodiments, in view of the present disclosure,any pharmaceutically acceptable carrier suitable for use in an antibodypharmaceutical composition can be used in the invention.

Pharmaceutically acceptable acidic/anionic salts for use in theinvention include, and are not limited to acetate, benzenesulfonate,benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate,carbonate, chloride, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, glyceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,lactobionate, malate, maleate, mandelate, mesylate, methylbromide,methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate,pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate,tosylate and triethiodide. Organic or inorganic acids also include, andare not limited to, hydriodic, perchloric, sulfuric, phosphoric,propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,saccharinic or trifluoroacetic acid.

Pharmaceutically acceptable basic/cationic salts include, and are notlimited to aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (alsoknown as tris(hydroxymethyl)aminomethane, tromethane or “TRIS”),ammonia, benzathine, t-butylamine, calcium, chloroprocaine, choline,cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine,magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium,procaine, quinine, sodium, triethanolamine, or zinc.

In some embodiments of the invention, pharmaceutical formulations areprovided comprising the conjugates of the invention in an amount fromabout 0.001 mg/ml to about 100 mg/ml, from about 0.01 mg/ml to about 50mg/ml, or from about 0.1 mg/ml to about 25 mg/ml. The pharmaceuticalformulation may have a pH from about 3.0 to about 10, for example fromabout 3 to about 7, or from about 5 to about 9. The formulation mayfurther comprise at least one ingredient selected from the groupconsisting of a buffer system, preservative(s), tonicity agent(s),chelating agent(s), stabilizer(s) and surfactant(s).

The formulation of pharmaceutically active ingredients withpharmaceutically acceptable carriers is known in the art, e.g.,Remington: The Science and Practice of Pharmacy (e.g. 21st edition(2005), and any later editions). Non-limiting examples of additionalingredients include: buffers, diluents, solvents, tonicity regulatingagents, preservatives, stabilizers, and chelating agents. One or morepharmaceutically acceptable carrier may be used in formulating thepharmaceutical compositions of the invention.

In one embodiment of the invention, the pharmaceutical composition is aliquid formulation. A preferred example of a liquid formulation is anaqueous formulation, i.e., a formulation comprising water. The liquidformulation may comprise a solution, a suspension, an emulsion, amicroemulsion, a gel, and the like. An aqueous formulation typicallycomprises at least 50% w/w water, or at least 60%, 70%, 75%, 80%, 85%,90%, or at least 95% w/w of water.

In one embodiment, the pharmaceutical composition may be formulated asan injectable which can be injected, for example, via a syringe or aninfusion pump. The injection may be delivered subcutaneously,intramuscularly, intraperitoneally, or intravenously, for example.

In another embodiment, the pharmaceutical composition is a solidformulation, e.g., a freeze-dried or spray-dried composition, which maybe used as is, or whereto the physician or the patient adds solvents,and/or diluents prior to use. Solid dosage forms may include tablets,such as compressed tablets, and/or coated tablets, and capsules (e.g.,hard or soft gelatin capsules). The pharmaceutical composition may alsobe in the form of sachets, dragees, powders, granules, lozenges, orpowders for reconstitution, for example.

The dosage forms may be immediate release, in which case they maycomprise a water-soluble or dispersible carrier, or they may be delayedrelease, sustained release, or modified release, in which case they maycomprise water-insoluble polymers that regulate the rate of dissolutionof the dosage form in the gastrointestinal tract.

In other embodiments, the pharmaceutical composition may be deliveredintranasally, intrabuccally, or sublingually.

The pH in an aqueous formulation can be between pH 3 and pH 10. In oneembodiment of the invention, the pH of the formulation is from about 7.0to about 9.5. In another embodiment of the invention, the pH of theformulation is from about 3.0 to about 7.0.

In another embodiment of the invention, the pharmaceutical compositioncomprises a buffer. Non-limiting examples of buffers include: arginine,aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaricacid, glycine, glycylglycine, histidine, lysine, maleic acid, malicacid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate,sodium phosphate, succinate, tartaric acid, tricine, andtris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffer maybe present individually or in the aggregate, in a concentration fromabout 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml toabout 20 mg/ml. Pharmaceutical compositions comprising each one of thesespecific buffers constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a preservative. Non-limiting examples of buffers include:benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine,chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate,imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol,2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate,thiomerosal, and mixtures thereof. The preservative may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificpreservatives constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises an isotonic agent. Non-limiting examples of the embodimentinclude a salt (such as sodium chloride), an amino acid (such asglycine, histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, and threonine), an alditol (such as glycerol,1,2-propanediol propyleneglycol), 1,3-propanediol, and 1,3-butanediol),polyethyleneglycol (e.g. PEG400), and mixtures thereof. Another exampleof an isotonic agent includes a sugar. Non-limiting examples of sugarsmay be mono-, di-, or polysaccharides, or water-soluble glucans,including for example fructose, glucose, mannose, sorbose, xylose,maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch,and sodium carboxymethylcellulose. Another example of an isotonic agentis a sugar alcohol, wherein the term “sugar alcohol” is defined as aC(4-8) hydrocarbon having at least one —OH group. Non-limiting examplesof sugar alcohols include mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, and arabitol. Pharmaceutical compositions comprisingeach isotonic agent listed in this paragraph constitute alternativeembodiments of the invention. The isotonic agent may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificisotonic agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a chelating agent. Non-limiting examples of chelating agentsinclude citric acid, aspartic acid, salts of ethylenediaminetetraaceticacid (EDTA), and mixtures thereof. The chelating agent may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificchelating agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a stabilizer. Non-limiting examples of stabilizers include oneor more aggregation inhibitors, one or more oxidation inhibitors, one ormore surfactants, and/or one or more protease inhibitors.

In another embodiment of the invention, the pharmaceutical compositioncomprises a stabilizer, wherein said stabilizer iscarboxy-/hydroxycellulose and derivates thereof (such as HPC, HPC-SL,HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol(such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone,salts (such as sodium chloride), sulphur-containing substances such asmonothioglycerol), or thioglycolic acid. The stabilizer may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificstabilizers constitute alternative embodiments of the invention.

In further embodiments of the invention, the pharmaceutical compositioncomprises one or more surfactants, preferably a surfactant, at least onesurfactant, or two different surfactants. The term “surfactant” refersto any molecules or ions that are comprised of a water-soluble(hydrophilic) part, and a fat-soluble (lipophilic) part. The surfactantmay, for example, be selected from the group consisting of anionicsurfactants, cationic surfactants, nonionic surfactants, and/orzwitterionic surfactants. The surfactant may be present individually orin the aggregate, in a concentration from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificsurfactants constitute alternative embodiments of the invention.

In a further embodiment of the invention, the pharmaceutical compositioncomprises one or more protease inhibitors, such as, e.g., EDTA(ethylenediamine tetraacetic acid), and/or benzamidine hydrochloric acid(HCl). The protease inhibitor may be present individually or in theaggregate, in a concentration from about 0.1 mg/ml to about 20 mg/ml.Pharmaceutical compositions comprising each one of these specificprotease inhibitors constitute alternative embodiments of the invention.

The pharmaceutical composition of the invention may comprise an amountof an amino acid base sufficient to decrease aggregate formation of thepolypeptide during storage of the composition. The term “amino acidbase” refers to one or more amino acids (such as methionine, histidine,imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan,threonine), or analogues thereof. Any amino acid may be present eitherin its free base form or in its salt form. Any stereoisomer (i.e., L, D,or a mixture thereof) of the amino acid base may be present. The aminoacid base may be present individually or in the combination with otheramino acid bases, in a concentration from about 0.01 mg/ml to about 50mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml.Pharmaceutical compositions comprising each one of these specific aminoacid bases constitute alternative embodiments of the invention.

It is also apparent to one skilled in the art that the therapeuticallyeffective dose for conjugates of the present invention or apharmaceutical composition thereof will vary according to the desiredeffect. Therefore, optimal dosages to be administered may be readilydetermined by one skilled in the art and will vary with the particularconjugate used, the mode of administration, the strength of thepreparation, and the advancement of the disease condition. In addition,factors associated with the particular subject being treated, includingsubject age, weight, diet and time of administration, will result in theneed to adjust the dose to an appropriate therapeutic level.

For all indications, the conjugates of the invention are preferablyadministered peripherally at a dose of about 1 μg to about 5 mg per dayin single or divided doses (e.g., a single dose can be divided into 2,3, 4, 5, 6, 7, 8, 9, or 10 subdoses), or at about 0.01 μg/kg to about500 μg/kg per dose, more preferably about 0.05 μg/kg to about 250 μg/kg,most preferably below about 50 μg/kg. Dosages in these ranges will varywith the potency of each agonist, of course, and are readily determinedby one of skill in the art. The above dosages are thus exemplary of theaverage case. There can, of course, be individual instances where higheror lower dosage ranges are merited, and such are within the scope ofthis invention.

In certain embodiments, the conjugates of the invention are administeredat a dose of about 1 μg to about 5 mg, or at a dose of about 0.01 μg/kgto about 500 μg/kg, more preferably at a dose of about 0.05 μg/kg toabout 250 μg/kg, most preferably at a dose below about 50 μg/kg with adose of a second therapeutic agent (e.g., liraglutide) at a dose ofabout 1 μg to about 5 mg, or at a dose of about 0.01 μg/kg to about 500μg/kg, more preferably at a dose of about 0.05 μg/kg to about 250 μg/kg,most preferably at a dose below about 50 μg/kg.

The pharmaceutically-acceptable salts of the conjugates of the inventioninclude the conventional non-toxic salts or the quaternary ammoniumsalts which are formed from inorganic or organic acids or bases.Examples of such acid addition salts include acetate, adipate, benzoate,benzenesulfonate, citrate, camphorate, dodecylsulfate, hydrochloride,hydrobromide, lactate, maleate, methanesulfonate, nitrate, oxalate,pivalate, propionate, succinate, sulfate and tartrate. Base saltsinclude ammonium salts, alkali metal salts such as sodium and potassiumsalts, alkaline earth metal salts such as calcium and magnesium salts,salts with organic bases such as dicyclohexylamino salts and salts withamino acids such as arginine. Also, the basic nitrogen-containing groupsmay be quaternized with, for example, alkyl halides.

The pharmaceutical compositions of the invention may be administered byany means that accomplish their intended purpose. Examples includeadministration by parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, transdermal, buccal or ocular routes. Administrationmay be by the oral route. Suitable formulations for parenteraladministration include aqueous solutions of the active conjugates inwater-soluble form, for example, water-soluble salts, acidic solutions,alkaline solutions, dextrose-water solutions, isotonic carbohydratesolutions and cyclodextrin inclusion complexes.

The present invention also encompasses a method of making apharmaceutical composition comprising mixing a pharmaceuticallyacceptable carrier with any of the conjugates of the present invention.Additionally, the present invention includes pharmaceutical compositionsmade by mixing one or more pharmaceutically acceptable carriers with anyof the conjugates of the present invention.

Furthermore, the conjugates of the present invention may have one ormore polymorph or amorphous crystalline forms and as such are intendedto be included in the scope of the invention. In addition, theconjugates may form solvates, for example with water (i.e., hydrates) orcommon organic solvents. As used herein, the term “solvate” means aphysical association of the conjugates of the present invention with oneor more solvent molecules. This physical association involves varyingdegrees of ionic and covalent bonding, including hydrogen bonding. Incertain instances the solvate will be capable of isolation, for examplewhen one or more solvent molecules are incorporated in the crystallattice of the crystalline solid. The term “solvate” is intended toencompass both solution-phase and isolatable solvates. Non-limitingexamples of suitable solvates include ethanolates, methanolates, and thelike.

It is intended that the present invention include within its scopepolymorphs and solvates of the conjugates of the present invention.Thus, in the methods of treatment of the present invention, the term“administering” shall encompass the means for treating, ameliorating orpreventing a syndrome, disorder or disease described herein with theconjugates of the present invention or a polymorph or solvate thereof,which would obviously be included within the scope of the inventionalbeit not specifically disclosed.

In another embodiment, the invention relates to the conjugates of theinvention for use as a medicament.

The present invention includes within its scope prodrugs of theconjugates of this invention. In general, such prodrugs will befunctional derivatives of the conjugates which are readily convertiblein vivo into the required conjugate. Thus, in the methods of treatmentof the present invention, the term “administering” shall encompass thetreatment of the various disorders described with the conjugatespecifically disclosed or with a conjugate which may not be specificallydisclosed, but which converts to the specified conjugate in vivo afteradministration to the patient. Conventional procedures for the selectionand preparation of suitable prodrug derivatives are described, forexample, in “Design of Prodrugs”, Ed. H. Bundgaard, Elsevier, 1985.

Furthermore, it is intended that within the scope of the presentinvention, any element, in particular when mentioned in relation to theconjugates of the invention, shall comprise all isotopes and isotopicmixtures of said element, either naturally occurring or syntheticallyproduced, either with natural abundance or in an isotopically enrichedform. For example, a reference to hydrogen includes within its scope ¹H,²H (D), and ³H (T). Similarly, references to carbon and oxygen includewithin their scope respectively 12C, ¹³C and ¹⁴C and ¹⁶O and ¹⁸O. Theisotopes may be radioactive or non-radioactive. Radiolabelled conjugatesof the invention may comprise a radioactive isotope selected from thegroup of ³H, ¹¹C, ¹⁸F, ¹²²I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and⁸²Br. Preferably, the radioactive isotope is selected from the group of³H, ¹¹C and ¹⁸F.

Some conjugates of the present invention may exist as atropisomers.Atropisomers are stereoisomers resulting from hindered rotation aboutsingle bonds where the steric strain barrier to rotation is high enoughto allow for the isolation of the conformers. It is to be understoodthat all such conformers and mixtures thereof are encompassed within thescope of the present invention.

Where the conjugates according to this invention have at least onestereo center, they may accordingly exist as enantiomers ordiastereomers. It is to be understood that all such isomers and mixturesthereof are encompassed within the scope of the present invention.

Where the processes for the preparation of the conjugates according tothe invention give rise to mixture of stereoisomers, these isomers maybe separated by conventional techniques such as preparativechromatography. The conjugates may be prepared in racemic form, orindividual enantiomers may be prepared either by enantiospecificsynthesis or by resolution. The conjugates may, for example, be resolvedinto their component enantiomers by standard techniques, such as theformation of diastereomeric pairs by salt formation with an opticallyactive acid, such as (−)-di-p-toluoyl-D-tartaric acid and/or(+)-di-p-toluoyl-L-tartaric acid followed by fractional crystallizationand regeneration of the free base. The conjugates may also be resolvedby formation of diastereomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the conjugates may be resolved using a chiral column viahigh performance liquid chromatography (HPLC) or SFC. In some instancesrotamers of conjugates may exist which are observable by 1H NMR leadingto complex multiplets and peak integration in the 1H NMR spectrum.

During any of the processes for preparation of the conjugates of thepresent invention, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This maybe achieved by means of conventional protecting groups, such as thosedescribed in Protective Groups in Organic Chemistry, ed. J. F. W.McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, John Wiley & Sons, 1991, each of which isherein incorporated by reference in its entirety for all purposes. Theprotecting groups may be removed at a convenient subsequent stage usingmethods known from the art.

Methods of Use

The present invention also provides a method for preventing, treating,delaying the onset of, or ameliorating a disorder, disease, or conditionor any one or more symptoms of said disorder, disease, or condition in asubject in need thereof, the method comprising administering to thesubject in need thereof an effective amount of a conjugate orpharmaceutical composition of the invention.

According to particular embodiments, the disease disorder, or conditioncan be any disease, disorder, or condition that could be treated with apeptide or compound capable of being coupled to the monoclonal antibodyplatform of the present invention. In certain embodiments, the disease,disorder or condition is selected from the group consisting of obesity,type I or II diabetes, metabolic syndrome (i.e., Syndrome X), insulinresistance, impaired glucose tolerance (e.g., glucose intolerance),hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia dueto congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis,diabetic nephropathy, and other cardiovascular risk factors such ashypertension and cardiovascular risk factors related to unmanagedcholesterol and/or lipid levels, osteoporosis, inflammation,non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH), renal disease, and/or eczema.

According to particular embodiments, a therapeutically effective amountrefers to the amount of therapy which is sufficient to achieve one, two,three, four, or more of the following effects: (i) reduce or amelioratethe severity of the disease, disorder or condition to be treated or asymptom associated therewith; (ii) reduce the duration of the disease,disorder or condition to be treated, or a symptom associated therewith;(iii) prevent the progression of the disease, disorder or condition tobe treated, or a symptom associated therewith; (iv) cause regression ofthe disease, disorder or condition to be treated, or a symptomassociated therewith; (v) prevent the development or onset of thedisease, disorder or condition to be treated, or a symptom associatedtherewith; (vi) prevent the recurrence of the disease, disorder orcondition to be treated, or a symptom associated therewith; (vii) reducehospitalization of a subject having the disease, disorder or conditionto be treated, or a symptom associated therewith; (viii) reducehospitalization length of a subject having the disease, disorder orcondition to be treated, or a symptom associated therewith; (ix)increase the survival of a subject with the disease, disorder orcondition to be treated, or a symptom associated therewith; (xi) inhibitor reduce the disease, disorder or condition to be treated, or a symptomassociated therewith in a subject; and/or (xii) enhance or improve theprophylactic or therapeutic effect(s) of another therapy.

The therapeutically effective amount or dosage can vary according tovarious factors, such as the disease, disorder or condition to betreated, the means of administration, the target site, the physiologicalstate of the subject (including, e.g., age, body weight, health),whether the subject is a human or an animal, other medicationsadministered, and whether the treatment is prophylactic or therapeutic.Treatment dosages are optimally titrated to optimize safety andefficacy.

As used herein, the terms “treat,” “treating,” and “treatment” are allintended to refer to an amelioration or reversal of at least onemeasurable physical parameter related the disease, disorder, orcondition, which is not necessarily discernible in the subject, but canbe discernible in the subject. The terms “treat,” “treating,” and“treatment,” can also refer to causing regression, preventing theprogression, or at least slowing down the progression of the disease,disorder, or condition. In a particular embodiment, “treat,” “treating,”and “treatment” refer to an alleviation, prevention of the developmentor onset, or reduction in the duration of one or more symptomsassociated with the disease, disorder, or condition. In a particularembodiment, “treat,” “treating,” and “treatment” refer to prevention ofthe recurrence of the disease, disorder, or condition. In a particularembodiment, “treat,” “treating,” and “treatment” refer to an increase inthe survival of a subject having the disease, disorder, or condition. Ina particular embodiment, “treat,” “treating,” and “treatment” refer toelimination of the disease, disorder, or condition in the subject.

In one embodiment, the invention provides a method for preventing,treating, delaying the onset of, or ameliorating obesity, or any one ormore symptoms of obesity in a subject in need thereof, the methodcomprising administering to the subject in need thereof an effectiveamount of a conjugate or pharmaceutical composition of the invention. Insome embodiments, the body weight of a subject is reduced, for example,by between about 0.01% to about 0.1%, between about 0.1% to about 0.5%,between about 0.5% to about 1%, between about 1% to about 5%, betweenabout 2% to about 3%, between about 5% to about 10%, between about 10%to about 15%, between about 15% to about 20%, between about 20% to about25%, between about 25% to about 30%, between about 30% to about 35%,between about 35% to about 40%, between about 40% to about 45%, orbetween about 45% to about 50%, relative to the body weight of a subjectprior to administration of any of the conjugates, pharmaceuticalcompositions, forms, or medicaments of the invention described herein,or compared to control subjects not receiving any of the conjugates,compositions, forms, medicaments, or combinations of the inventiondescribed herein.

In some embodiments, the reduction in body weight is maintained forabout 1 week, for about 2 weeks, for about 3 weeks, for about 1 month,for about 2 months, for about 3 months, for about 4 months, for about 5months, for about 6 months, for about 7 months, for about 8 months, forabout 9 months, for about 10 months, for about 11 months, for about 1year, for about 1.5 years, for about 2 years, for about 2.5 years, forabout 3 years, for about 3.5 years, for about 4 years, for about 4.5years, for about 5 years, for about 6 years, for about 7 years, forabout 8 years, for about 9 years, for about 10 years, for about 15years, or for about 20 years, for example.

The present invention provides a method of preventing, treating,delaying the onset of, or ameliorating a syndrome, disorder or disease,or any one or more symptoms of said syndrome, disorder, or disease in asubject in need thereof, wherein said syndrome, disorder or disease isselected from the group consisting of obesity, type I or type IIdiabetes, metabolic syndrome (i.e., Syndrome X), insulin resistance,impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia,hyperinsulinemia, hypertriglyceridemia, dyslipidemia, atherosclerosis,diabetic nephropathy, and other cardiovascular risk factors such ashypertension and cardiovascular risk factors related to unmanagedcholesterol and/or lipid levels, osteoporosis, inflammation,non-alcoholic steatohepatitis (NASH), renal disease, and eczema,comprising administering to the subject in need thereof an effectiveamount of a conjugate or pharmaceutical composition of the invention.

As used herein, metabolic syndrome refers to a subject having any one ormore of the following: high blood sugar (e.g., high fasting bloodsugar), high blood pressure, abnormal cholesterol levels (e.g., low HDLlevels), abnormal triglyceride levels (e.g., high triglycerides), alarge waistline (i.e., waist circumference), increased fat in theabdominal area, insulin resistance, glucose intolerance, elevatedC-reactive protein levels (i.e., a proinflammatory state), and increasedplasma plasminogen activator inhibitor-1 and fibrinogen levels (i.e., aprothrombotic state).

The present invention provides a method of reducing food intake in asubject in need thereof, the method comprising administering to thesubject in need thereof an effective amount of a conjugate orpharmaceutical composition of the invention. In some embodiments, foodintake of a subject is reduced, for example, by between about 0.01% toabout 0.1%, between about 0.1% to about 0.5%, between about 0.5% toabout 1%, between about 1% to about 5%, between about 2% to about 3%,between about 5% to about 10%, between about 10% to about 15%, betweenabout 15% to about 20%, between about 20% to about 25%, between about25% to about 30%, between about 30% to about 35%, between about 35% toabout 40%, between about 40% to about 45%, or between about 45% to about50%, relative to food intake of a subject prior to administration of anyof the conjugates, compositions, forms, medicaments, or combinations ofthe invention described herein, or compared to control subjects notreceiving any of the conjugates, compositions, forms, medicaments, orcombinations of the invention described herein.

In some embodiments, the reduction in food intake is maintained forabout 1 week, for about 2 weeks, for about 3 weeks, for about 1 month,for about 2 months, for about 3 months, for about 4 months, for about 5months, for about 6 months, for about 7 months, for about 8 months, forabout 9 months, for about 10 months, for about 11 months, for about 1year, for about 1.5 years, for about 2 years, for about 2.5 years, forabout 3 years, for about 3.5 years, for about 4 years, for about 4.5years, for about 5 years, for about 6 years, for about 7 years, forabout 8 years, for about 9 years, for about 10 years, for about 15years, or for about 20 years, for example.

The present invention provides a method of reducing glycated hemoglobin(A1C) in a subject in need thereof, the method comprising administeringto the subject in need thereof an effective amount of a conjugate orpharmaceutical composition of the invention. In some embodiments, A1C ofa subject is reduced, for example, by between about 0.001% and about0.01%, between about 0.01% and about 0.1%, between about 0.1% and about0.2%, between about 0.2% and about 0.3%, between about 0.3% and about0.4%, between about 0.4% and about 0.5%, between about 0.5% and about1%, between about 1% and about 1.5%, between about 1.5% and about 2%,between about 2% and about 2.5%, between about 2.5% and about 3%,between about 3% and about 4%, between about 4% and about 5%, betweenabout 5% and about 6%, between about 6% and about 7%, between about 7%and about 8%, between about 8% and about 9%, or between about 9% andabout 10% relative to the A1C of a subject prior to administration ofany of the conjugates, compositions, forms, medicaments, or combinationsof the invention described herein, or compared to control subjects notreceiving any of the conjugates, compositions, forms, medicaments, orcombinations of the invention described herein.

In other embodiments, methods are provided for reducing fasting bloodglucose levels in a subject in need thereof, the methods comprisingadministering to the subject in need thereof an effective amount of aconjugate or pharmaceutical composition of the invention. Fasting bloodglucose levels may be reduced to less than about 140 to about 150 mg/dL,less than about 140 to about 130 mg/dL, less than about 130 to about 120mg/dL, less than about 120 to about 110 mg/dL, less than about 110 toabout 100 mg/dL, less than about 100 to about 90 mg/dL, or less thanabout 90 to about 80 mg/dL, relative to the fasting blood glucose levelsof a subject prior to administration of any of the conjugates,compositions, forms, medicaments, or combinations of the inventiondescribed herein, or compared to control subjects not receiving any ofthe conjugates, compositions, forms, medicaments, or combinations of theinvention described herein.

The present invention provides a method of modulating Y2 receptoractivity in a subject in need thereof, the method comprisingadministering to the subject in need thereof an effective amount of aconjugate or pharmaceutical composition of the invention. As usedherein, “modulating” refers to increasing or decreasing receptoractivity.

In some embodiments, an effective amount of a conjugate of the inventionor a form, composition or medicament thereof is administered to asubject in need thereof once daily, twice daily, three times daily, fourtimes daily, five times daily, six times daily, seven times daily, oreight times daily. In other embodiments, an effective amount of aconjugate of the invention or a form, composition or medicament thereofis administered to a subject in need thereof once every other day, onceper week, twice per week, three times per week, four times per week,five times per week, six times per week, two times per month, threetimes per month, or four times per month.

Another embodiment of the invention comprises a method of preventing,treating, delaying the onset of, or ameliorating a disease, disorder orsyndrome, or one or more symptoms of any of said diseases, disorders, orsyndromes in a subject in need thereof, the method comprisingadministering to the subject in need thereof an effective amount of aconjugate or pharmaceutical composition of the invention in acombination therapy. In certain embodiments, the combination therapy isa second therapeutic agent. In certain embodiments, the combinationtherapy is a surgical therapy.

As used herein, the term “in combination,” in the context of theadministration of two or more therapies to a subject, refers to the useof more than one therapy.

As used herein, combination therapy refers to administering to a subjectin need thereof one or more additional therapeutic agents, or one ormore surgical therapies, concurrently with an effective amount of aconjugate of the invention or a form, composition or medicament thereof.In some embodiments, the one or more additional therapeutic agents orsurgical therapies can be administered on the same day as an effectiveamount of a conjugate of the invention, and in other embodiments, theone or more additional therapeutic agents or surgical therapies may beadministered in the same week or the same month as an effective amountof a conjugate of the invention.

In certain embodiments, wherein the disease or disorder is selected fromthe group consisting of obesity, type II diabetes, metabolic syndrome,insulin resistance and dyslipidemia, the second therapeutic agent can bean antidiabetic agent. In certain embodiments, the antidiabetic agentcan be a glucagon-like peptide-1 (GLP-1) receptor modulator.

The present invention also contemplates preventing, treating, delayingthe onset of, or ameliorating any of the diseases, disorders, syndromes,or symptoms described herein in a subject in need thereof with acombination therapy that comprises administering to the subject in needthereof an effective amount of a conjugate or pharmaceutical compositionof the invention, in combination with any one or more of the followingtherapeutic agents: a dipeptidyl peptidase-4 (DPP-4) inhibitor (e.g.,sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); a GLP-1receptor agonist (e.g., short-acting GLP-1 receptor agonists such asexenatide and lixisenatide; intermediate-acting GLP-1 receptor agonistssuch as liraglutide; long-acting GLP-1 receptor agonists such asexenatide extended-release, albiglutide, dulaglutide); a sodium-glucoseco-transporter-2 (SGLT-2) inhibitors (e.g., canaglifozin, dapaglifozin,empaglifozin, etc.); bile acid sequestrants (e.g., colesevelam, etc.);dopamine receptor agonists (e.g., bromocriptine quick-release);biguanides (e.g., metformin, etc.); insulin; oxyntomodulin;sulfonylureas (e.g., chlorpropamide, glimepiride, glipizide, glyburide,glibenclamide, glibornuride, glisoxepide, glyclopyramide, tolazamide,tolbutamide, acetohexamide, carbutamide, etc.); and thiazolidinediones(e.g; pioglitazone, rosiglitazone, lobeglitazone, ciglitazone,darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone,etc.). In some embodiments, the dose of the additional therapeuticagent(s) is reduced when given in combination with a conjugate of theinvention. In some embodiments, when used in combination with aconjugate of the invention, the additional therapeutic agent(s) may beused in lower doses than when each is used singly.

In certain embodiments, wherein the disease or disorder is selected fromthe group consisting of obesity, type I or type II diabetes, metabolicsyndrome (i.e., Syndrome X), insulin resistance, impaired glucosetolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia,hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism(CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and othercardiovascular risk factors such as hypertension and cardiovascular riskfactors related to unmanaged cholesterol and/or lipid levels,osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), renal disease, and eczema, thesecond therapeutic agent can be liraglutide.

The present invention contemplates preventing, treating, delaying theonset of, or ameliorating any of the diseases, disorders, syndromes, orsymptoms described herein in a subject in need thereof, with acombination therapy that comprises administering to the subject in needthereof an effective amount of a conjugate or pharmaceutical compositionof the invention in combination with a surgical therapy. In certainembodiments, the surgical therapy can be bariatric surgery (e.g.,gastric bypass surgery, such as Roux-en-Y gastric bypass surgery; sleevegastrectomy; adjustable gastric band surgery; biliopancreatic diversionwith duodenal switch; intragastric balloon; gastric plication; andcombinations thereof).

In embodiments in which the one or more additional therapeutic agents isadministered on the same day as an effective amount of a conjugate ofthe invention, the conjugate of the invention may be administered priorto, after, or simultaneously with the additional therapeutic agent. Theuse of the term “in combination” does not restrict the order in whichtherapies are administered to a subject. For example, a first therapy(e.g., a composition described herein) can be administered prior to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after)the administration of a second therapy to a subject.

Embodiments

The invention provides also the following non-limiting embodiments.

Embodiment 1 is an isolated antibody or antigen binding fragment thereofcomprising a light chain variable region having completely human Iggermline V gene sequences, and a heavy chain variable region havingcompletely human Ig germline V gene sequences except HCDR3 having theamino acid sequence of SEQ ID NO:18, wherein the antibody or antigenbinding fragment thereof does not specifically bind to any human antigenin vivo.

Embodiment 2 is the isolated monoclonal antibody or antigen-bindingfragment thereof of embodiment 1, wherein the isolated monoclonalantibody or antigen-binding fragment thereof comprises a heavy chaincomplementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a lightchain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3,having the polypeptide sequences of SEQ ID NO: 16, 17, 18, 19, 20, and21, respectively

Embodiment 3 is the isolated monoclonal antibody or antigen-bindingfragment thereof of embodiment 2, wherein the isolated monoclonalantibody comprises a heavy chain variable domain (VH) having thepolypeptide sequence of SEQ ID NO:12, and a light chain variable domain(VL) having the polypeptide sequence of SEQ ID NO:14.

Embodiment 4 is the isolated monoclonal antibody of any one ofembodiments 1-3, further comprising a Fc portion.

Embodiment 5 is the isolated monoclonal antibody of embodiment 4,wherein the Fc portion further comprises a Fc region derived from humanIgG4 Fc region.

Embodiment 6 is the isolated monoclonal antibody of embodiment 5,wherein the human IgG4 Fc region has substitutions that eliminateeffector function.

Embodiment 7 is the isolated monoclonal antibody of embodiment 6,wherein the monoclonal antibody further comprises a modified human IgG4Fc region containing at least one substitution selected from the groupconsisting of a proline for glutamate at residue 233, an alanine orvaline for phenylalanine at residue 234, an alanine or glutamate forleucine at residue 235, an alanine for asparagine at residue 297.

Embodiment 8 is the isolated monoclonal antibody of embodiment 6 or 7,wherein the human IgG4 Fc region comprises a substitution of a serine toproline at position 228.

Embodiment 9 is the isolated monoclonal antibody of any one ofembodiments 4-8, comprising a heavy chain (HC) having the polypeptidesequence of SEQ ID NO:13, and a light chain (LC) having the polypeptidesequence of SEQ ID NO:15.

Embodiment 10 is an isolated nucleic acid encoding the monoclonalantibody or antigen binding fragment thereof of any one of embodiments1-9.

Embodiment 11 is a vector comprising the isolated nucleic acid ofembodiment 10.

Embodiment 12 is a host cell comprising the vector of embodiment 11.

Embodiment 13 is a method of producing an isolated monoclonal antibodyor antigen binding fragment thereof, the method comprising culturing thehost cell of embodiment 12 under conditions to produce the monoclonalantibody or antigen binding fragment thereof, and recovering theantibody or antigen-binding fragment thereof from the cell or culture.

Embodiment 14 is the isolated monoclonal antibody or antigen-bindingfragment thereof of any one of embodiments 1-9, further comprising atleast one pharmacologically active moiety conjugated thereto.

Embodiment 15 is the isolated monoclonal antibody or antigen-bindingfragment thereof of embodiment 14, wherein the pharmacologically activemoiety is a therapeutic peptide.

Embodiment 16 is the isolated monoclonal antibody or antigen-bindingfragment thereof of embodiment 15, wherein the therapeutic peptide isconjugated at the cysteine residue of SEQ ID NO:18.

Embodiment 17 is the isolated monoclonal antibody or antigen-bindingfragment thereof of embodiment 15 or 16, wherein the therapeutic peptideis conjugated to the antibody or antigen-binding fragment thereof via alinker.

Embodiment 18 is the isolated monoclonal antibody or antigen-bindingfragment thereof of embodiment 17, wherein the linker comprises apeptide linker, a hydrocarbon linker, a polyethylene glycol (PEG)linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, apolyester linker, or a hybrid linker consisting of PEG and an embeddedheterocycle.

Embodiment 19 is the isolated monoclonal antibody or antigen-bindingfragment thereof of any one of embodiments 15-18, wherein thetherapeutic peptide is selected from the group consisting ofoxyntomodulin, glucagon-like peptide 1 (GLP1), peptide tyrosine tyrosine(PYY), exendin (exenatide), amylin (pramlintide), alpha-melanocytestimulating hormone (MSH), cocaine- and amphetamine-regulated transcript(CART), neuropeptide Y receptor Y1 (NPY1) antagonists, neuropeptide Yreceptor Y5 (NPY5) antagonists, neurotensin S, neuropeptide B,neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3), galanin,cholecystokinin (CCK), orexin, melanin-concentrating hormone (MCH),oxytocin, and stresscopin.

Embodiment 20 is the isolated monoclonal antibody or antigen bindingfragment thereof of embodiment 19, wherein the therapeutic peptide isoxyntomodulin comprising the polypeptide sequence of SEQ ID NO:24.

Embodiment 21 is a conjugate comprising a monoclonal antibody or antigenbinding fragment thereof coupled to an oxyntomodulin therapeuticpeptide, wherein the conjugate has the structure of SEQ ID NO:27 asshown in FIG. 11, wherein mAb represents a monoclonal antibody orantigen binding fragment thereof according to any one of embodiments1-9, and ]₂ represents that 1 or 2 of the oxyntomodulin therapeuticpeptide are covalently conjugated to the mAb.

Embodiment 22 is a method of producing the isolated monoclonal antibodyor antigen-binding fragment thereof of any one of embodiments 15-21,comprising reacting an electrophile, preferably bromoacetamide ormaleimide, introduced onto a sidechain of the therapeutic peptide, withthe sulfhydryl group of the cysteine residue of SEQ ID NO:18 of themonoclonal antibody or antigen-binding fragment thereof, therebycreating a covalent linkage between the therapeutic peptide and themonoclonal antibody or antigen-binding fragment thereof.

Embodiment 23 is a pharmaceutical composition comprising the isolatedmonoclonal antibody or antigen-binding fragment thereof of any one ofembodiments 14-21 and a pharmaceutically acceptable carrier.

Embodiment 24 is a method of producing a pharmaceutical compositioncomprising the monoclonal antibody or antigen-binding fragment thereofof any one of embodiments 14-21, the method comprising combining themonoclonal antibody or antigen-binding fragment thereof with apharmaceutically acceptable carrier to obtain the pharmaceuticalcomposition.

Embodiment 25 is a kit comprising the monoclonal antibody orantigen-binding fragment thereof of any one of embodiments 1-9 and14-21.

Embodiment 26 is a method of increasing the half-life of a therapeuticpeptide in a subject, the method comprising conjugating the therapeuticpeptide with a monoclonal antibody or antigen-binding fragment thereofcomprising a heavy chain complementarity determining region 1 (HCDR1),HCDR2, HCDR3, and a light chain complementarity determining region 1(LCDR1), LCDR2, and LCDR3, having the polypeptide sequences of SEQ IDNO: 16, 17, 18, 19, 20, and 21, respectively, wherein the therapeuticpeptide is conjugated to the monoclonal antibody or antigen-bindingfragment thereof at the Cys residue of SEQ ID NO:18.

Embodiment 27 is the method of embodiment 26, wherein the therapeuticpeptide is selected from the group consisting of oxyntomodulin,glucagon-like peptide 1 (GLP1), peptide tyrosine tyrosine (PYY), exendin(exenatide), amylin (pramlintide), alpha-melanocyte stimulating hormone(MSH), cocaine- and amphetamine-regulated transcript (CART),neuropeptide Y receptor Y1 (NPY1) antagnoists, neuropeptide Y receptorY5 (NPY5) antagonists, neurotensin S, neuropeptide B, neuropeptide W,ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystokinin(CCK), orexin, melanin-concentrating hormone (MCH), oxytocin, andstresscopin.

Embodiment 28 is the method of embodiment 27, wherein the therapeuticpeptide is oxyntomodulin.

Embodiment 29 is the method of claim 28, wherein the oxyntomodulin has apolypeptide sequence of SEQ ID NO:24.

Embodiment 30 is the method of any one of embodiments 25-29, wherein themonoclonal antibody comprises a heavy chain variable domain (VH) havingthe polypeptide sequence of SEQ ID NO:12.

Embodiment 31 is the method of any one of claims 25-30, wherein themonoclonal antibody comprises a heavy chain (HC) having the polypeptidesequence of SEQ ID NO:13.

Embodiment 32 is the method of any one of embodiments 25-31, wherein themonoclonal antibody comprises a light chain variable domain (VL) havingthe polypeptide sequence of SEQ ID NO:14.

Embodiment 33 is the method of any one of embodiments 25-32, wherein themonoclonal antibody comprises a light chain (LC) having the polypeptidesequence of SEQ ID NO:15.

Embodiment 34 is an isolated monoclonal antibody or antigen-bindingfragment thereof comprising a heavy chain complementarity determiningregion 3 comprising SEQ ID NO:18, wherein the isolated monoclonalantibody or antigen-binding fragment thereof is capable of being coupledto a therapeutic peptide.

EXAMPLES Example 1: Identification and Production of mAb MSCB97Selection of PH9L3 VL and PH9115 VH as Starting V Regions forEngineering

The antibody light chain variable region (VL) designated PH9L3 (SEQ IDNO:3) (Teplyakov et al., “Structural diversity in a human antibodygermline library,” mAbs August-September 8(6):1045-63 (2016)) and theantibody heavy chain variable region (VH) designated PH9H5 (SEQ ID NO:4)(Teplyakov et al., “Structural diversity in a human antibody germlinelibrary,” mAbs August-September 8(6):1045-63 (2016)) were selected asthe starting variable regions from which to engineer a mAb enabled forpeptide conjugation. PH9L3 is comprised completely of human Ig germlineV gene sequences and as such does not contain any sequence mutationsfrom the in vivo affinity maturation process that would result in highaffinity, antigen-specific binding. The CDR3 of PH9H5 is the onlysegment not comprised of human germline V gene sequences in that VH.CDR3 (SEQ ID NO:5) of PH9H5 is identical to the CDR3 of an anti-humanCCL2 antibody, CNTO 888, a neutralizing CCL2 antibody, see, e.g., US20100074886 A1, the relevant disclosure on CNTO 888 is incorporated byreference in its entirety. A Fab containing the PH9H5/PH9L3 VH/VL pairwas generated.

The PH9L3, PH9H5 and the human Ig germline V-region and J-regionsequences to which these are most similar were aligned to determinesequence identity or similarity to the germline sequences. PH9H5 wasaligned to a concatenation (SEQ ID NO:6) of human Ig germline genesIGHV3-23*01 (PubMed ID: M99660) (SEQ ID NO:7) and human IGHJ1*01 (PubMedID: J00256) (SEQ ID NO:8), with the only difference between the PH9H5amino acid sequence and the concatenated human IGHV3-23*01-IGHJ1*01sequence being at VH CDR3, which was SEQ ID NO:5 for PH9H5.

PH9L3 was aligned to a concatenation (SEQ ID NO:9) of human Ig germlinegenes IGKV3-11*01 (PubMed ID: X01668) (SEQ ID NO:10) and IGKJ1*01(PubMed ID: J00242) (SEQ ID NO:11), with the only difference being onedeviation in the V gene/J gene junction.

Design and Generation of Cys Substituted Variants of PH9H5 and PH9L3

Variants of the PH9H5 VH that contain a single Cys substitution atselect CDR residues across all three CDRs of the V region were designed,generated and cloned as complete heavy chains with a human IgG1 constantregion into a mammalian host expression vector. The PH9H5/PH9L3 Fabstructure was utilized to aid in selection of CDR residues forsubstitution that appear more accessible for conjugation and in some ofthe variants, additional glycine (Gly) residues were inserted on eitherside of the introduced Cys residue to potentially increase accessibilityof the Cys for conjugation. Similar variants of the PH9L3 VL weredesigned and generated except these were cloned as complete light chainswith a human kappa constant region into the expression vector. A totalof 24 expression constructs of PH9H5 single Cys variants and 22expression constructs of PH9L3 single Cys variants were generated. Theresidues selected for substitution within PH9H5_VH (SEQ ID NO:4) andwithin PH9L3_VL (SEQ ID NO:3) are summarized in FIG. 2.

The expression constructs generated were used to express the Cysvariants by transiently co-transfecting each PH9H5 based HC Cys variantconstruct with the wild type PH9L3 LC construct or co-transfecting eachPH9L3 based LC Cys variant construct with the wild type PH9H5 HCconstruct. Initial test transfections used HEK-derived Expi293 asexpression host and were at 20 ml scale. The majority of both HC and LCCys variants expressed well based on variant protein quantitation fromculture supernatant.

Five initial HC Cys variants, MSCB33-MSCB37, were expressed in Expi293at 750 ml scale and variant proteins purified. The purification yieldand quality properties of the purified variants were fairly similar andsufficient for using the purified proteins in initial peptideconjugation reactions.

Evaluation of Peptide Conjugation to PH9115-Based HC Cys Variants

Analytical mass determination of MSCB33 protein and other variantproteins indicated the presence of cysteine adducts at the Cysengineered for conjugation, with two per mAb, as well as removal of theHC C-terminal Lys residue, which is commonly seen in recombinantlyproduced mAbs. To prepare the variant mAbs for conjugation, adducts wereremoved by a reduction process developed to maintain the nativedisulfide bonds within the mAb (see Example 3). Initial testconjugations with a human oxyntomodulin (OXM) peptide analogue (GCGAib2, Gly16,24, Arg20, Leu27, Lys30 (PEG₁₂)-NH₂) were done usingmaleimide chemistry on all five HC Cys variant mAbs. Conjugationefficiency differed between mAb variants, which was qualitativelyestimated by the conjugation reaction products and the relativepercentage of each. Greatest efficiency, as measured by greatestpercentage of homodimer product, was observed with MSCB33, as comparedto the other I102C variants containing flanking Gly residues, and littleor no conjugation was observed with the Y103C variants MSCB35 or MSCB37.

Several other HC and LC Cys variants, with engineered cysteinesubstitutions in various CDRs (a T28C, S30C, and S54C substitution inPH9H5_VH (SEQID NO:129), and an S30C and S92C substitution in PHpL3_VL(SEQ ID NO:128)) were expressed at large scale in Expi293. Removal ofthe Cys adducts from these purified proteins by reduction waschallenging and these variants were not pursued further. Due to thechallenges observed with most of the Cys variants and the initial goodconjugation efficiency observed with the I102C PH9H5 variant mAb,MSCB33, process development and further engineering efforts focused onthis particular variant.

Fc Engineering of MSCB33

MSCB33 was re-engineered to contain the silent, human IgG4_PAA Fc toreduce Fc function in vivo. Human IgG4_PAA has mutationsS228P/F234A/L235A on the human IgG4 allotype nG4m(a) (based on IGHG4*01allele as defined in IMGT). An expression construct with the VH ofMSCB33 fused to the IgG4_PAA Fc was generated and used together with thesame LC expression construct used for MSCB33 expression to produce theIgG4_PAA variant of MSCB33, which is designated MSCB97. The amino acidsequences of MSCB97 VH, HC, VL, and LC are provided by SEQ ID NO:12, 13,14, and 15, respectively.

Test expression of MSCB97 was done transiently at 20 ml scale in Expi293cells and this mAb variant expressed well. MSCB97 was purified from alarge scale, Expi293 expression run. The purification yield of MSCB97was 264.53 mg/L, and the quality was determined at 85% monomer species.Subsequent largescale expression runs and purifications were similar orbetter in yield and quality and indicated the consistency with whichthis mAb could be produced.

Evaluation of Peptide Conjugation to MSCB97 and Conjugation ReactionScalability

The LC-HC disulfide connectivity differs between the IgG1 and IgG4isotypes, so the reduction and maleimide conjugation was tested andconfirmed to be translatable from the IgG1 mAb MSCB33 to the IgG4_PAAmAb MSCB97 using TCEP reduction and conjugation of the OXM-maleimidetest peptide described above. The linkage resulting from maleimideconjugation is known to be potentially reversible, so bromoacetamideconjugation chemistry, which produces a more stable linkage, was adoptedand implemented successfully on a 10 mg scale based on starting MSCB97.

The MSCB97 conjugate of the OXM analogue GCG Aib2, Glu16,24, Arg20,Leu27, Lys30-ε-(PEG₁₂)-NH₂ (compound 2—“compound 2” and “conjugate 2”can be used interchangeably herein) (MSCB97 conjugated toH-Aib-QGTFTSDYSKYLDERRARDFVEWLLNTK-(COCH₂CH₂(OCH₂CH₂)₁₂NHCOCH₂Br)—NH₂(SEQ ID NO: 24) to form compound 2 (FIG. 11; SEQ ID NO:27)) generatedthrough bromoacetamide chemistry was assayed to determine in vitroGLP-1R and GCGR potencies. Potencies relative to reference peptides andreference unstructured peptide conjugates were reasonable and similar tothat of the conjugate generated with the same peptide to MSCB33 (IgG1).This demonstrated that the single difference between MSCB97 (IgG4_PAA)and MSCB33 (IgG1), which is isotype, had no impact on the potency ofconjugates containing the same peptide. Additionally, these data showedthat desirable in vitro potencies could be retained in a peptide-mAbconjugate generated with bromoacetamide chemistry that produces alinkage that is stable in vivo. Other OXM analogues were also conjugatedto MSCB97 and in vitro potencies of these conjugates assayed. Theseconjugates had similar GLP-1R and GCGR potencies to compound 2,highlighting the ability to conjugate a variety of peptides to MSCB97,while retaining peptide potency.

Evaluation of Peptide-MSCB97 Conjugate Binding to Human CCL2

While MSCB97 was selected and engineered for lack of specific antigenbinding, the most likely antigen that this mAb might bind, if any, ishuman CCL2 based on the origin of the VH CDR3. Whether MSCB97 doesdemonstrate any specific CCL2 binding was evaluated using twopeptide-MSCB97 conjugates, with an OXM peptide analogue, (compound 2),or a PYY peptide analogue, (compound 1—“compound 1” and “conjugate 1”can be used interchangeably herein).

Potential CCL2 binding was directly measured by surface plasmonresonance (SPR) in which the conjugates were surface-immobilized usingan anti-Fc capture method. A commercially available anti-CCL2 mouse mAbserved as a positive control and two non-specific human antibodies, CNTO9412 and HH3B33, served as negative controls. All controls weresimilarly surface-immobilized and recombinant human CCL2 was flowed overimmobilized conjugates and controls at concentrations up to 400 nM.Based on the pre-established assay criteria, CCL2 accumulation,indicating specific antigen binding, was seen with the positive controlbut not with the negative controls nor with either peptide-MSCB97conjugate (compound 1 or 2). This confirmed that MSCB97, in the relevanttherapeutic form of a peptide-mAb conjugate, lacks human CCL2 binding.

SPR binding method: Binding measurements using Surface Plasmon Resonance(SPR) were performed using a ProteOn XPR36 system (BioRad). A biosensorsurface was prepared by coupling a mixture of anti-Human IgG Fc (Jacksoncat#109-005-098) and anti-Mouse IgG Fc (Jackson cat#315-005-046) to themodified alginate polymer layer surface of a GLC chip (BioRad,Cat#176-5011) using the manufacturer instructions for amine-couplingchemistry. Approximately 5700 RU (response units) of mAbs wereimmobilized. The binding experiments were performed at 25° C. in runningbuffer (DPBS; 0.01% P20; 100 μg/ml BSA). To perform binding kineticexperiment, samples (compound 2, compound 1, and control mAbs-positiveand negative) were captured followed by injections of Recombinant humanCCL2 (Thermo, catalog #RMCP120) at 5 concentrations (in a 4-fold serialdilution). The association phase was monitored for 3 minutes at 50μL/min, then followed by 5 minutes of buffer flow (dissociation phase).The chip surface was regenerated with two 18 second pulses of 100 mMH₃PO₄ (Sigma, Cat#7961) at 100 μL/min.

The collected data were processed using ProteOn Manager software. First,the data was corrected for background using inter-spots. Then, doublereference subtraction of the data was performed by using the bufferinjection for analyte injections. Pre-established assay criteria fordetermining specific detectable binding of compound 2, compound 1, andthe positive control to CCL2 required a dose proportional responsewith >10 RU signal at the highest concentration, and negative controlresponse signals<10 RU. Based on the assay criteria, the results foreach sample was reported as Yes or No to dose-response binding to humanCCL2.

Example 2: Expression and Purification of the mAb

The fully human monoclonal antibody (mAb) can be recombinantly expressedin a mammalian expression host and purified from the cell culturesupernatant using standard methods that are known in the field. Forexample, a cDNA sequence encoding the light (LC) and heavy chains (HC)of the mAb, each including an appropriate signal peptide to enablesecretion, can be cloned into separate mammalian expression vectors orinto a single expression vector using standard molecular biologymethods. Expression vectors used can be any of those commerciallyavailable such as pEE12.4, pcDNA™3.1(+) or pIRESpuro3 or any customexpression vector with similar functionalities. In such vectorstranscription of the heavy and light chains of the mAb are each drivenby any of the known effective promoters such as the hCMV-MIE promoter.Transfection grade plasmid DNA is prepared for separate LC and HCexpression constructs or a single construct expressing both LC and HCusing standard methods such as a QIAGEN Plasmid Midi Kit.

Purified plasmid DNA is prepared for transfection with a lipid-basedtransfection reagent such as Freestyle™ Max transfection reagent,following manufacturer's instructions, and is then transfected into astandard mammalian expression host cell line, such as CHO-S or HEK293-F. If the mAb LC and HC are encoded by separate expressionconstructs, the two constructs are simultaneously transfected. Prior toand after transfection, mammalian cells are cultured for maintenance orfor mAb expression following standard cell culture methods whereby thecell density ranges to maintain, the culture media to use, and the othercell culture conditions followed are determined by the specificmammalian host cell line utilized. These parameters are typicallydocumented by the vendor from which the cell line was obtained or in thescientific literature. For example, CHO-S cells are maintained in CHOFreestyle™ media in suspension, shaking at 125 RPM in a humidifiedincubator set at 37° C. and 8% CO₂, and split when the cellconcentration is between 1.5 and 2.0×10⁶ cells per ml.

Cell culture supernatants from the transiently transfected mammaliancells expressing the mAb are harvested several days after transfection,clarified by centrifugation and filtered. Duration of expression forCHO-S cells is typically four days but can be adjusted and can differfor different mammalian host cell lines. Large scale transfections (>10liters) are concentrated 10-fold using a concentrator such as aCentramate. The mAb is purified from the clarified supernatant using aProtein A affinity column such as the HiTrap Mab Select Sure utilizingstandard methods for binding mAb to Protein A resin, washing the resinand eluting the protein using low pH buffer. The protein fractions areneutralized immediately by elution into tubes containing pH 7 buffer andpeak fractions are pooled, filtered and dialyzed against phosphatebuffered saline (PBS), pH 7.2 overnight at 4° C. After dialysis the mAbis filtered again (0.2μ filter) and the protein concentration isdetermined by absorbance at 280 nm. Quality of the purified mAb proteinis assessed by SDS-PAGE (polyacrylamide gel electrophoresis) andanalytical size exclusion HPLC and endotoxin levels are measured using alimulus amebocyte lysate (LAL) assay. Purified mAb is stored at 4° C.

Expression and Purification of MSCB97 from Transiently Transfected CHOCells

MSCB97 was expressed in ExpiCHO-S™ cells (ThermoFisher Scientific,Waltham, Mass.; Cat # A29127) by transient transfection of the cellswith purified plasmid DNA of a MSCB97 expression construct followingmanufacturer's recommendations. Briefly, ExpiCHO-S™ cells weremaintained in suspension in ExpiCHO™ expression medium (ThermoFisherScientific, Cat # A29100) in a shaking incubator set at 37° C., 8% CO₂and 125 RPM. The cells were passaged so that on the day of transfection,dilution down to 6.0×10⁶ cells per ml could be achieved, maintainingcell viability at 98% or better. Transient transfections were done usingthe ExpiFectamine™ CHO transfection kit (ThermoFisher Scientific Cat #A29131). For each ml of diluted cells to be transfected, one microgramof plasmid DNA is used and diluted into OptiPRO™ SFM complexationmedium. ExpiFectamine™ CHO reagent is used at a 1:3 ratio (v/v,DNA:reagent) and also diluted into OptiPRO™. The diluted DNA andtransfection reagent were combined for one minute, allowing DNA/lipidcomplex formation, and then added to the cells. After overnightincubation, ExpiCHO™ feed and ExpiFectamine™ CHO enhancer were added tothe cells. Cells were cultured with shaking at 32° C. for five daysprior to harvesting the culture supernatants.

Culture supernatants from the transiently transfected ExpiCHO-S™ cellswere harvested by clarifying through centrifugation (30 min, 6000 rpm)followed by filtration (0.2μ PES membrane, Corning). Large scaletransfections (5 to 20 liters) were first concentrated 10-fold using aPall Centramate Tangential Flow Filtration system. 10× Dulbecco'sphosphate-buffered saline (DPBS), pH7.2 was added to the supernatant to1× final concentration prior to loading onto an equilibrated (DPBS, pH7.2) HiTrap Mab Select Sure Protein A column (GE Healthcare; LittleChalfont, United Kingdom) at a relative concentration of ˜20 mg proteinper ml of resin, using an AKTA FPLC chromatography system. Afterloading, the column was washed with 10 column volumes of DPBS, pH7.2.The protein was eluted with 10 column volumes of 0.1 M Na-Acetate, pH3.5. Protein fractions were neutralized immediately by elution intotubes containing 2.0 M tris(hydroxymethyl)aminomethane (Tris), pH 7 at20% the elution fraction volume. Peak fractions were pooled and the pHadjusted to −5.5 with additional Tris, if necessary. The purifiedprotein was filtered (0.2μ) and the concentration was determined byabsorbance at 280 nm on a BioTek SynergyHT™ spectrophotometer. Thequality of the purified protein was assessed by SDS-PAGE and analyticalsize exclusion HPLC (Dionex HPLC system). The endotoxin level wasmeasured using a turbidometric LAL assay (Pyrotell®-T, Associates ofCape Cod).

Example 3: Conjugation of mAb and Cyclic PYY Peptides

Method A: Partial Reduction of mAb with TCEP

A 10 mg/mL solution of mAb in tris-acetate buffer (20 mL, 1 mM in EDTA)was treated with 3 equivalents of TCEP. The solution was adjusted to pH6 and after 1 hr at room temperature (rt) high pressure liquidchromatography with mass spectrometer (LCMS) showed that the disulfideadducts at position C102 had been completely reduced. The reduced mAbwas purified by protein A adsorption and elution (4 CV 100 mM aceticacid) to provide 180 mg of reduced mAb.

Conjugation of Reduced mAb and Cyclic PYY Peptides

Lyophilized peptide (5 eq vs mAb) was added to the reduced mAb describedabove. EDTA was added to a final concentration of 1 mM and the pH wasadjusted to 7. The concentration was adjusted to 8 mg/mL and thereaction was allowed to proceed with gentle agitation for 16 h at roomtemperature. TCEP (0.5 eq vs mAb) was added and the reaction was allowedto proceed further for 4 hr at rt with gentle agitation, after whichtime the high molecular weight (MW) species were reduced to less than3%.

The reaction mixture was adjusted to pH 5.5 and purified by ion exchangechromatography on CaptoSP resin using a gradient 100% A (100 mMTRIS-acetate, pH 5.5) to 100% B (100 mM TRIS-acetate, pH 5.5; 0.5 MNaCl) over 20 CV. Fractions containing the desired conjugate were pooledand 140 mg of conjugate were recovered, coeluting with a small amount ofunreacted peptide. Final purification was by protein A adsorption andelution (4 CV 100 mM acetic acid). The pH of the product was adjusted to6 to give 120 mg of conjugate (60% yield) at >90% purity with <3% highMW species.

Method B

Hydrophobic Interaction Chromatography (HIC) Purification of mAb

A 20 mg/mL solution of mAb in tris-acetate buffer was loaded on ahydrophobic interaction column (TOSOH TSKgel phenyl 7.5×21 cm) andeluted with a linear gradient (0-70% B/A, solvent A: 5% iPrOH, 1M(NH₄)₂SO₄, 100 mM phosphate buffer, pH 6.0; solvent B: 20% iPrOH, 100 mMphosphate buffer). The mAb monomer peaks were pooled, concentrated (5-10mg/mL) and dialyzed against 3-(N-morpholino) propanesulfonic acid (MOPS)buffer (100 mM, pH 5.5).

Partial Reduction with TCEP and Conjugation of Reduced mAb with PeptideAnalog

To the purified mAb (27 mL, 9.28 mg/mL) was added 4 eq. TCEP followed byEDTA (1 mM). After 2 hr at room temperature LCMS showed that thedisulfide adducts at position C102 had been completely reduced. Thereduced mAb was treated with Zebra desalting spin column (7×10 mL, 7KMWCO, pre-equilibrium with MOPS 100 mM pH 5.5) to remove the liberatedcysteines/GSH. To the combined fractions of the reduced mAb (28 mL) wasadded a solution of cyclic PYY peptide in Milli Q grade water (6.5 eq vsmAb, 15-20 mg/mL) followed by EDTA (1 mM). The pH of the reaction wasadjusted to 7.2 to 7.4 by dropwise addition of 1N NaOH. The reaction wasallowed to proceed 18 h at room temperature with gentle agitation. Thereaction was continued for another 12 h after addition of a further 0.5equiv TCEP to reduce mAb-mAb dimer formed during the course of thereaction and allow conversion to the desired mAb homodimer. The pH ofthe reaction was lowered to pH 5.5 by addition of 2 M acetic acid andthe crude conjugate was purified by hydrophobic interactionchromatography and eluted with a linear gradient (0-100% B/A, solvent A:5% iPrOH, 1M (NH₄)2SO4, 100 mM phosphate buffer, pH 6.0; solvent B: 20%iPrOH, 100 mM phosphate buffer). Final purification was by protein Aadsorption (PBS) and elution (NaOAc, pH 3.5). The pH of the product wasadjusted to 6 and dialyzed against PBS to give the final sample (56%).

Alternatively, the mAb was reduced with GSH and/or Cys. After removal ofthe reducing agent by Tangential Flow Filtration (TFF), an excess of thepeptide was added to the reduced mAb optionally in the presence of0.2-0.5 equivalents of TCEP.

Example 4: In Vitro Studies

Compound 1 (SEQ ID NO:2), a monoclonal antibody couple to a cyclic PYYpeptide (FIG. 3), was evaluated for its ability to activate NPYreceptors in vitro in clonal cells (HEK or CHO) expressing human, rat,mouse and rhesus monkey Y2 receptors, and human Y1, Y4 and Y5 receptors.PYY₃₋₃₆, NPY and PP were included in these assays as study controls.

Cell Lines

Stable transfected clonal cell lines expressing NPY receptors weredeveloped for use in cAMP assays. In brief, HEK293 cell lines weretransfected using the Lipofectamine 2000 kit (Invitrogen) according toits protocol with expression plasmids carrying the coding sequences forthe human Y2 receptor (Accession No.: NM_000910.2), the human Y5receptor (Accession No.: NM_006174.2), the mouse Y2 receptor (AccessionNo.: NM_008731) and the rhesus monkey Y2 receptor (Accession No.:NM_001032832). Forty eight hours after transfection, cells werere-plated with selection media (DMEM high glucose with 10% fetal bovineserum (FBS), 50 I.U. penicillin, 50 μg/ml streptomycin, 2 mML-glutamine, 1 mM sodium pyruvate and 600 μg/ml G418). Cells were keptin selection media for 2 weeks before single clones were picked usinglimited dilution method. The transfected cells were subsequentlymaintained by culturing in DMEM-high glucose media (Cellgro)supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% sodiumpyruvate, 1% penicillin/streptomycin and 600 μg/ml G418.

In addition, CHO-K1 cell lines were obtained from DiscoverX Corporation,expressing the human Y1 receptor (Catalog No.: 93-0397C2) and the humanY4 receptor (Catalog No.: 95-0087C2). The DiscoverX cells cultured inF12 media (Gibco) supplemented with 10% FBS and under the G418 selection(800 μg/mL). The rat Y2 receptor was expressed in a Glo-Sensor CHO-K1line obtained from Promega Corporation. These cells were transfectedwith the pGloSensor™-23F cAMP plasmid for a luminescent based cAMP assaybut had been tested and validated for use with the Perkin-Elmer LANCEcAMP assay. The rat Y2 cells were grown in F12 media (Gibco)supplemented with 10% FBS and 800 μg/ml G418.

All the cell lines were banked in vials (4×10⁶ cells/vial) and stored inliquid nitrogen until use. The day before the assay, the vials werethawed and added to 15 mls of appropriate media. Cells were centrifugedat 450×g for 5 min, supernatants were aspirated and cells werere-suspended in media without G418 at a density of 0.2×10⁶ cells/ml.Cells were dispensed (25 μl/well) into Biocoat collagen-coated white 384well plates to a final density of 5000 cells/well. The cell plates wereincubated overnight in a 37° C. humidified tissue culture incubatorunder 5% CO₂/90% O₂ atmosphere.

Experimental Protocol

The cAMP assay was the same for the various receptor assays. The LANCEcAMP kit (Perkin Elmer Corporation; Waltham, Mass.) was used in allexperiments to quantitate intracellular cAMP levels. On the day of theassay, the cell media was decanted from the cells and 6 μl of peptides(2× concentration) was added to the wells. Peptides were made up as an11 point dose response (starting at 100 nM or 10 μM with serial 1:3dilutions) in stimulation buffer. Stimulation buffer consists of 5 mMHEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 500 μM IBMXand 0.1% bovine serum albumin (BSA) in HBSS (Hank's balanced saltsolution). Next 6 μl of stimulation buffer containing forskolin (2×, 5μM final concentration) and LANCE cAMP antibody (1:100) was added to thecells. After incubation for 25 minutes at rt, 12 μl of assay detectionmix was added to each well. The detection mix was prepared by dilutingbiotin-cAMP (1:750) and Europium-W8044 (1:2250) in detection buffer asprovided with the LANCE cAMP kit. The plate was incubated for 2 hours atrt and then read as a TR-FRET assay on the Envision plate reader(excitation 320 nm, emission 615 nm and 665 nm). Channel 1 fluorescence(relative fluorescent units at 615 nm) and channel 2 fluorescence(relative fluorescent units at 665 nm) along with their ratio wereexported into an Excel file.

Data Analysis

Data from the Envision plate reader were expressed as relativefluorescence units (RFU) calculated as (615 nm/665 nm)×10,000. Allsamples were measured in triplicate. Data were analyzed using theCrucible in-house data analysis software, designed by Eudean Shaw. Theunknown cAMP concentrations within each well were interpolated from thereference standards of known cAMP concentrations included within eachplate. Parameters such as EC₅₀, Log(EC₅₀), HillSlope (nH), top, andbottom, were derived by plotting cAMP concentration values over logcompound concentrations fitted with 4-P model using a non-linearweighted least squares application within R environment (Open Sourcehttp://cran.us.r-project.org/) implemented by the Non-ClinicalStatistics & Computing department at Janssen R&D.

TABLE 1 in vitro Data Cross-species potency (EC₅₀ nM) Human isoformpotency Rhesus (EC₅₀ nM) Mouse Rat Monkey Compound hY2 hY1 hY4 hY5 Y2 Y2Y2 Compound 1 0.006 >2910 >2910 345.7 0.004 0.04 0.004 PYY3-36 0.08 66.4136.7 12.3 0.03 0.50 0.06

Example 5: Pharmacokinetics (PK)

DIO Mouse PK

Male DIO C57BL/6N mice (20 weeks of age, 14 weeks on high fat diet) wereobtained from Taconic Laboratory. Mice were housed one mouse per cagewith AlphaDri bedding in a temperature-controlled room with 12-hlight/dark cycle. Mice were allowed ad libitum access to water andmaintained on high fat diet (D12492, Research Diet).

Mice were dosed subcutaneously (s.c.) with 1 mg/kg compound 1, 3 animalswere sacrificed at each time point and blood was collected at t=4, 8,24, 48, 72, 120, and 168 hours. Blood from 3 naïve animals was alsocollected. Approximately 300 μL of blood from each animal was collectedvia jugular vein after decapitation while under gas anesthesia inducedwith 70% CO₂ and 30% O₂ mixture. Blood samples (approximately 300 μL)was collected in K3E (EDTA) coated Sarstedt Microvette® tubes containing12 μl (4% ratio) of complete protease inhibitor solution and 3 μL (1%ratio) of DPP-IV inhibitor. Blood samples were placed on wet ice priorto being centrifuged at 10,000 rpm for ˜4 minutes under refrigeratedconditions (˜5° C.) for cell removal within 30 minutes followingcollection at each time point and all available plasma was transferredto a 96-well plate. The well plate was stored on dry ice until it wasplaced in a −80° C. freezer. Data are shown in Table 2 and FIG. 4.

Rat PK

Compound 1 was administered subcutaneously and intravenously to maleSprague-Dawley rats (Charles River Laboratories, Wilmington, Mass.) at adose level of 1.0 mg/kg in PBS, (pH 7.0-7.6). Approximately 500 μL ofblood was collected from three animals per time point via a saphenousvein (t=1, 4, 24, 48, 72, 96, 168, and 240 hours post-dose). A 336 hourpost-dose blood sample was collected via jugular vein after decapitationwhile under gas anesthesia induced with 70% CO₂ and 30% O₂ mixture.Blood samples were collected in K3E (EDTA) coated Sarstedt Microvette®tubes containing 20 μl (4% ratio) of complete protease inhibitorsolution and 5 μL (1% ratio) of DPPIV inhibitor. Blood samples wereplaced on wet ice prior to being centrifuged at 10,000 rpm for ˜4minutes under refrigerated conditions (˜5° C.) for cell removal within60 minutes following collection at each time point and all availableplasma was transferred to a 96-well plate. The levels of Compound 1 weremeasured using the LCMS method described below. Data are shown in Table3.

Cynomolgus Monkey (Cyno) PK

All animals were fasted for at least eight hours prior to dosing andthrough the first four hours of blood sample collection. Three animalsreceived a single IV dose of 1 mg/kg Compound 1 and three animalsreceived a single SC dose of 1 mg/kg Compound 1. Blood was collectedpre-dose and at 1, 6, 10, 24, 36, 48, 72, 120, 168, 240, 336, 432, and504 hours post-dose. An additional sample was collected at 0.5 hourspost-dose for the IV group. Approximately 1 ml of blood from each animalwas collected in K3E (EDTA) coated Sarstedt Microvette® tubes containing4% ratio of complete protease inhibitor solution and 1% ratio of DPPIVinhibitor. Blood samples were placed on wet ice prior to beingcentrifuged within 30 minutes following collection at each time pointand the resulting plasma was split in thirds and transferred intotriplicate 96-well plate. The well plate was stored on dry ice until itwas placed in a −80° C. freezer. Data are shown in Table 4 and FIG. 5.

Intact Mass Spec Assay for Determination of Plasma Levels

Plasma samples were processed by immuno-affinity capture using ananti-human Fc antibody, followed by reversed phase LC-high resolutionfull scan MS analysis on a triple TOF (time-of-flight) massspectrometer. The raw MS spectra were deconvoluted to elucidate themolecular weights of the components in the injected samples. The peak ofthe molecule ion of the intact conjugate was used for quantitation.Standard curve and quality control samples were prepared by spiking thereference standard in plasma and processed using the same procedure atthe same time as the incurred samples. PK data for DIO mouse, rat, andcyno are shown in Tables 2-4, respectively. PK data for DIO mouse andCyno are also shown in FIGS. 4 and 5, respectively.

TABLE 2 PK in DIO Mouse Dose T_(1/2) T_(max) C_(max) AUC_(last) Assay(mg/kg) hr hr ng/mL hr*ng/mL Intact 1.0 81.05 48 8750 995460

TABLE 3 PK in rat Dose T_(1/2) T_(max) C_(max) AUC_(last) Route (mg/kg)hr hr ng/mL hr*ng/mL IV 1.0 93.0 1 19.5 809.2 SC 1.0 88.7 48 4.2 602.0

TABLE 4 PK in Cyno Dose T_(1/2) T_(max) C_(max) AUC_(last) Route (mg/kg)hr hr ng/mL hr*ng/mL IV 1.0 178.39 0.67 30290 1207.39 SC 1.0 104.32 105590 712.19

Example 6: Efficacy Studies In Vivo

Weight Loss in Diet-Induced Obese (DIO) Mice: Acute Dosing

Compound 1 was evaluated for its ability to reduce foot intake and bodyweight in male DIO C57Bl/6 mice after a single dose. Male DIO C57BL/6Nmice (20 weeks of age, 14 weeks on high fat diet) were obtained fromTaconic Laboratory. Mice were housed one mouse per cage with AlphaDribedding in a temperature-controlled room with 12-h light/dark cycle.Mice were allowed ad libitum access to water and maintained on high fatdiet (D12492, Research Diet). Animals were acclimated to the facilityfor at least one week prior to the start of the experiment.

The day prior to dosing, mice were grouped into cohorts of eight animalsbased on individual body weights. At 3:00-4:00 pm the following day,animals were weighed and treated with vehicle (dPBS, pH 7.2), Compound 1at a dose of 0.1, 0.3, 1.0, 3.0, or 7.5 nmol/kg, or Dulaglutide at 0.3nmol/kg via subcutaneous (s.c.) administration. Body weights and foodintake were measured 24 h, 48 h and 72 h after dosing and thepercentages of weight loss and reduction in food intake were calculated.Statistical analyses were performed using two-way repeated measuresANOVA with Tukey's post-test in Prism. All data are presented as themean±SEM (FIG. 6 and FIG. 7).

Weight Loss in Diet-Induced Obese Mice: Chronic Dosing

Compound 1 was evaluated for its ability to reduce foot intake and bodyweight and improve glucose homeostasis on repeat dosing in male DIOC57Bl/6 mice over a period of 8 days. Male DIO C57BL/6N mice (20 weeksof age, 14 weeks on high fat diet) were obtained from TaconicLaboratory. Mice were housed one mouse per cage with AlphaDri bedding ina temperature-controlled room with 12-h light/dark cycle. Mice wereallowed ad libitum access to water and maintained on high fat diet(D12492, Research Diet). Animals were acclimated to the facility for atleast one week prior to the start of the experiment.

The day prior to dosing, mice were grouped based on individual bodyweights. At 3:00-4:00 pm for each of the next 8 days, animals and foodintake were weighed. Animals were treated with vehicle (dPBS, pH 7.2) ordulaglutide at 0.3 nmol/kg via subcutaneous administration every day orCompound 1 at doses of 0.1, 0.3, 1.0, 3.0 nmol/kg via subcutaneousadministration every three days. After 8 days, mice are fasted for 5hours and then given a 2 g/kg bolus of glucose orally at t=0. At t=0,30, 60, 90, and 120 minutes post glucose challenge blood glucose ismeasured and at t=0, 30, and 90 minutes blood in drawn to measure plasmainsulin. Statistical analyses were performed using one-way ANOVA ortwo-way repeated measures ANOVA with Tukey's post-test in Prism. Alldata are presented as the mean±SEM (FIGS. 8 and 9, and Tables 5-8).

TABLE 5 Effect of compound 1 on body weight (g) over 8 days treatmentDulaglutide Treatment Vehicle Compound 1 (nmol/kg) (nmol/kg) Day n/a 0.10.3 1.0 3.0 0.3 −1 46.3 ± 0.6 46.3 ± 0.6 46.7 ± 0.8 46.3 ± 0.6 46.3 ±0.6 46.3 ± 0.6 0 46.1 ± 0.7 46.0 ± 0.6 46.8 ± 0.7 46.3 ± 0.6 46.3 ± 0.645.9 ± 0.6 1 45.8 ± 0.7 45.0 ± 0.6 45.2 ± 0.7 43.9 ± 0.5 43.9 ± 0.6 44.1± 0.6 2 45.2 ± 0.7 43.9 ± 0.6 43.6 ± 0.7 42.4 ± 0.7* 41.8 ± 0.7* 42.2 ±0.6* 3 45.1 ± 0.7 43.9 ± 0.6 42.8 ± 0.6 41.0 ± 0.6* 40.1 ± 0.6* 40.6 ±0.6* 4 44.7 ± 0.7 43.4 ± 0.6 42.0 ± 0.6 39.9 ± 0.6* 38.5 ± 0.6* 39.3 ±0.6* 5 44.5 ± 0.7 43.0 ± 0.6 41.5 ± 0.5* 39.1 ± 0.6* 36.8 ± 0.7* 38.1 ±0.7* 6 44.5 ± 0.7 43.1 ± 0.6 41.4 ± 0.5* 38.6 ± 0.6* 35.5 ± 0.8* 37.1 ±0.8* 7 44.3 ± 0.7 43.1 ± 0.7 41.3 ± 0.5* 38.2 ± 0.6* 34.8 ± 0.9* 36.4 ±1.0* 8 45.2 ± 0.6 46.3 ± 0.7 41.9 ± 0.5* 38.8 ± 0.6 34.7 ± 1.0* 36.5 ±1.1* Values represent mean ± SEM for data from 8 animals per time pergroup *p < 0.05, versus vehicle; two-way ANOVA RM, Tukey's multiplecomparison test

TABLE 6 Effect of compound 1 on blood glucose (mg/dL) levels during anOGTT after 8 days of treatment Delta Total AUC AUC Dose Time afterglucose challenge (min) (mg/dl/ (mg/dl/ Treatment (nmol/kg) 0 30 60 90120 120 min) 120 min) Vehicle NA 148 ± 6 227 ± 15 272 ± 12 206 ± 9 209 ±13 26492 ± 1232 8702 ± 1082 Comp. 1 0.1 151 ± 4 213 ± 9 249 ± 10 186 ±11 194 ± 15 24598 ± 849 6433 ± 677 0.3 137 ± 6 191 ± 11 208 ± 10* 146 ±8* 160 ± 8* 20786 ± 728* 4379 ± 364* 1.0 117 ± 5 146 ± 9* 203 ± 10* 135± 8* 136 ± 5* 18285 ± 769* 4319 ± 522* 3.0  73 ± 11* 129 ± 5* 149 ± 10* 93 ± 9* 101 ± 9* 13703 ± 913* 5004 ± 585 Dulaglutide 0.3  76 ± 9* 151 ±14* 174 ± 17* 104 ± 10* 119 ± 9* 15758 ± 1054* 6668 ± 1846 Valuesrepresent mean ± SEM for data from 8 animals per time per group *p <0.05, versus vehicle; two-way ANOVA RM, Tukey's multiple comparison testfor glucose values; one-way ANOVA. Tukey's multiple comparison test forAUC

TABLE 7 Effect of compound 1 on insulin (ng/mL) levels during an OGTTafter 8 days of treatment Dose Time after glucose challenge (min) TotalAUC Treatment (nmol/kg) 0 30 90 (mg/dl/120 min) Vehicle NA 5.6 ± 1.4 11.3 ± 3.0  4.0 ± 0.6 711.4 ± 164.1 Comp. 1 0.1 3.0 ± 0.4  6.6 ± 0.8*2.4 ± 0.2 415.6 ± 44.2  0.3 2.3 ± 0.2  4.0 ± 0.6* 1.7 ± 0.2 264.3 ±33.0* 1.0 1.3 ± 0.2* 2.2 ± 0.1* 1.1 ± 0.2 151.6 ± 12.5* 3.0 0.4 ± 0.1*1.2 ± 0.1*  0.4 ± 0.1* 73.9 ± 9.0* Dulaglutide 0.3 0.6 ± 0.1* 1.7 ± 0.2*0.7 ± 0.2 108.8 ± 12.3* Values represent mean ± SEM for data from 8animals per time per group *p < 0.05, versus vehicle; two-way ANOVA RM,Tukey's multiple comparison test for glucose values; one-way ANOVA,Tukey's multiple comparison test for AUC

TABLE 8 Effect of compound 1 on fed blood glucose (mg/dL) levels after 8days of treatment Treatment Dose (nmol/kg) Blood Glucose (mg/dL) VehicleNA 180 ± 5 Compound 1 0.1 164 ± 6 0.3 160 ± 5 1.0 149 ± 6 3.0  105 ± 12*Dulaglutide 0.3  114 ± 13* Values represent mean ± SEM for data from 8animals per time per group *p < 0.05, versus vehicle; one-way ANOVA,Tukey's multiple comparison test

Example 7: Synthetic Strategy for Preparation of mAb-Oxyntomodulin(mAb-OXM) Compound

Human oxyntomodulin (OXM) is a 37 amino-acid endogenous peptide (SEQ IDNO: 23) that has been shown to have beneficial pharmacology in patientswith type 2 diabetes mellitus. Pharmacology is the result of agonism atboth GLP1R and GCGR. Investigations of OXM analogs revealed a peptidevariant that demonstrated good glucose control and weight-loss in arodent model. The half-life of OXM in vivo is on the order of minutes inhumans. Additional design for OXM was therefore undertaken to confer ahalf-life commensurate with once-weekly dosing. An increase in half-lifewas accomplished by increasing the stability of the OXM peptide towardsproteolysis, and by increasing the circulating half-life of the peptideby covalent attachment of a monoclonal antibody (mAb). Proteolysis ofthe peptide payload by DPP4 was mitigated by substituting the serine atposition 2 with aminoisobutyric acid (Aib). The helical topology of thepeptide was stabilized by introducing a bifurcated salt bridge from Q20Rto S16E and Q24E, and a potential oxidation liability was mitigated bythe M27L variation. The parent mAb, MSCB97 (described above), was chosento have low intrinsic antigen binding, and an isoleucine to cysteinepoint mutation in the HCDR3 (SEQ ID NO:18) region of the heavy chain(I102C) served as the point of attachment for the synthetic peptidepayload. Effector function was silenced by using the IgG4 PAA isotype. Ashort oligoethylene glycol spacer was incorporated between the mAb andthe peptide to ensure unhindered access of the peptide to GLP1R andGCGR. The spacer also confers favorable aqueous solubility thatfacilitates the conjugation chemistry. Attachment of the peptide-spacerto the mAb was accomplished by introducing a reactive bromoacetamidegroup at the distal end of the glycol spacer. The proximal end of thespacer was attached to the OXM variant via the side-chain of K30. Theamino acid sequence of glucagon and the OXM peptide variant is providedby SEQ ID NOs:22 and 24, respectively. Conjugation was accomplished byreaction with the thiol functionality of the cysteine point mutation inthe mAb heavy chain. The amino acid sequences of the mAb heavy and lightchains are provided by SEQ ID NOs: 13 and 15, respectively.

Chemical Synthesis of mAb-OXM Compound

The overall synthetic scheme for the production of the mAb-OXM compoundis shown in FIG. 10.

Preparation of Oxyntomodulin (OXM) Peptide Variant:

The resin-bound protected peptide was synthesized using standard9-fluorenylmethyloxycarbonyl (Fmoc) amino acids (with the exception ofthe N-terminal His, Nα-Boc-His(Boc)-OH and Lys30, Nα-Fmoc-Lys(ivDDE)-OH)on PAL-PEG-polystyrene resin. Standard amino acid activation and Fmocdeprotection strategies were used throughout. Upon completion of thesequence, the side-chain of the C-terminal lysine was selectivelydeprotected by treatment with dilute anhydrous hydrazine inN,N-dimethylformamide (DMF). Fmoc-dPEG₁₂-CO₂H was coupled to theliberated amine using diisopropylcarbodiimide(DIC)/ethyl(hydroxyimino)-cyanoacetate activation. The Fmoc group wasremoved and the resulting amine was bromoacetylated using a solution ofbromoacetic anhydride in DMF.

The peptide was removed from the resin with simultaneous deprotection ofall the protecting groups by treatment with trifluoroacetic acid (TFA)containing phenol, water and triisopropylsilane as scavengers. The crudepeptide was isolated by cold precipitation with ether and purified byreverse phase high performance liquid chromatography (RP-HPLC) usingacetonitrile/water with 0.1% v/v TFA as eluent. The pure peptide wascollected as a fluffy white solid after lyophilization and stored at−80° C.

Reduction of Monoclonal Antibody MSCB97:

The monoclonal antibody is isolated with the engineered Cys102 residuedisulfide bonded to adventitious cysteine or glutathione residuesscavenged either from the cytosol or from the growth medium. Apreliminary reduction of these disulfides followed by removal of theunwanted cysteine is therefore required prior to conjugation of the OXMpeptide variant. Reduction was accomplished with the mAb immobilized onprotein A resin beads. Reductant (tricarboxyethyl phosphine, TCEP) wascirculated through the column at pH 5 until reduction was complete (1hr). The advantage of TCEP as reductant under these conditions is thatwhile reduction is effective, reformation of the disulfide bond at thelower pH is negligible. After washing away the by-products, the reducedadsorbed protein was eluted from the column using an acidic buffer(sodium acetate at pH 3.5). The reduced mAb was dialyzed twice against50 mM 3-(N-morpholino) propanesulfonic acid (MOPS) at pH 5.5

Smaller exploratory batches were prepared by reduction with 2.5equivalents of TCEP vs. mAb in solution at pH 5. The reduction wasallowed to proceed 2 hours at room temperature. Small moleculeby-products were removed by gel filtration (PD10 column) and the reducedMSCB97 was eluted with 10 mM MOPS, pH 5.5.

Preparation of mAb-OXM Compound:

A solution of reduced MSCB97 was added to a 7.6-fold excess oflyophilized OXM peptide variant. 1 mM EDTA solution was added to protectthe reactive thiols against metal-catalyzed oxidation, and the pH of thereaction solution was raised to 7.3 by addition of MOPS buffer (1 M, pH8.1). The reaction was allowed to proceed 18 h at room temperature withgentle agitation. The reaction was quenched by adjustment to pH 5.5 byaddition of 2 M acetic acid and the crude conjugate was purified byadsorption on protein A with washing to remove excess peptide, unfoldedproduct and by-products. Elution of the product (sodium acetate, pH 3.6)was followed by dialysis into acetate buffer pH 5.

Three independent syntheses were performed starting with 250 mg, 500 mgand 500 mg of MSCB97, and the products were combined into a singlebatch.

Analysis of mAb-OXM Compound

The properties of the prepared mAb-OXM compound were analyzed using (i)analytical hydrophobic interaction chromatography (HIC), (ii) intactmass measurement by liquid chromatography electrospray ionization massspectrometry (LC-ESIMS), (iii) analytical size-exclusion chromatography(SEC), and (iv) SDS-polyacrylamide gel electrophoresis.

Stability in Human and Monkey Plasma

OXM is metabolized rapidly by plasma peptidases. To determine theresistance of the mAb-OXM compound (“compound 2”) (FIG. 11) to enzymaticdegradation in human and monkey plasma, the ex vivo stability ofcompound 2 was evaluated. Compound 2 was incubated at 20 nM with fresh(never frozen) heparinized plasma at 37° C. for up to 168 hours.Bioanalysis was conducted using a functional cell-based bioassaymeasuring cAMP production in human GLP-1 receptor transfected HEK cells.The amount of cAMP produced was directly proportional to theconcentration of the active OXM analog, and levels of active OXM analogsin the stability samples were determined in this assay by interpolatingfrom reference standards of known concentrations. This assay was usedfor ranking of stability relative to the previously determined morestable control, control 1, and the previously determined less stablecontrol, control 2. The data was used to rank relative stability, andare shown in FIGS. 12 and 13.

Example 8: In Vitro Studies: In Vitro Potency of Compound 2 at theHuman, Mouse, Rat, and Cynomolgus Monkey GLP1 and Glucagon Receptors

The potency and species specificity of compounds of interest, includingcompound 2, were characterized in assays using HEK293 cells transfectedto stably express either human, cynomolgus monkey, rat or mouse GLP1 orGCG receptors. For each receptor assay, the clonal cells were seededinto 384-well plates at the appropriate densities ranging from 2000-5000cells/per well. Compounds were diluted in HBSS supplemented with 5 mMHEPES, 0.1% BSA and 0.5 mM IBMX and added to the cells. The cell plateswere incubated at room temperature for 5 or 10 minutes depending on thecell clone and then lysed for cAMP measurement. cAMP concentrations werequantitated using LANCE cAMP kits with an EnVision plate reader.Standard curves were included in each assay plate for back calculationof cAMP concentrations. Dose-response curves were analyzed and compoundEC₅₀ values calculated with Graphpad Prism or Crucible. The EC₅₀ dataare summarized in Tables 9-10.

TABLE 9 Summary of EC₅₀ values (nM) from human GLP1 and glucagonreceptor cAMP assays. Human GCGR GLP1R Glucagon 0.05 ± 0.01 0.96 ± 0.21Ser-8 GLP1 >100 0.07 ± 0.01 OXM 3.01 ± 0.35 2.40 ± 0.59 Compound 2 3.31± 0.22 0.43 ± 0.02 Dulaglutide >100 0.17 ± 0.04 Values represent mean ±SEM from 3-7 experiments.

TABLE 10 Summary of EC₅₀ values (nM) from mouse, rat, and cynomolgusmonkey GLP1 and glucagon receptor cAMP assays. Mouse Rat CynomolgusMonkey GCGR GLP1R GCGR GLP1R GCGR GLP1R Glucagon 0.09 ± 0.01 2.13 ± 0.421.57 ± 0.33 4.73 ± 0.20  0.03 ± 0.002 1.96 ± 0.38 Ser-8 GLP1 >100  0.06± 0.005 >100 0.14 ± 0.03 >100  0.08 ± 0.002 OXM 6.31 ± 2.58 1.90 ± 0.3112.74 ± 3.54  0.98 ± 0.16 0.49 ± 0.08 0.80 ± 0.07 Compound 2 0.82 ± 0.380.11 ± 0.02 5.90 ± 1.46 0.97 ± 0.13 2.03 ± 0.14 0.40 ± 0.03Dulaglutide >100 0.035 ± 0.002 >100 0.13 ± 0.03 >100 0.18 ± 0.01 Valuesrepresent mean ± SEM from 3-8 experiments.

Example 9: Potency at Other Related GPCRs

The potency of compound 2 was evaluated in assays using cells expressingseveral class B GPCRs. Compound 2 was tested in a panel of six in vitrocAMP assays using cells expressing CALCR, PTHR1, PTHR2, CRHR1, CRHR2 andVIPR1. The assays were performed according to the vendor's standardoperating procedures with a positive control included for each receptortested.

Additionally, compound 2 was tested in a stable cell line expressing thehuman GIP receptor. The cells were seeded into 384-well plates andtreated 24 hours later with the compounds for 5 minutes at roomtemperature. cAMP concentrations were measured using a CisBio HTRF cAMPkit and the results analyzed using GraphPad Prism. The data are shown inTables 11-12.

TABLE 11 Summary of compound 2 potency in cAMP assays. Compound TargetRC₅₀ (μM) Hill Curve Bottom Curve Top Max response Calcitonin CALCR0.0007764259 1.5254 −6.2187 105 101.04 PTH(1-34) PTHR1 0.0010801651.8672 −5.9767 101 93.574 Sauvagine CRHR1 0.0003082264 1.8133 −3.2011102.19 106.16 Sauvagine CRHR2 0.003395158 1.3095 −1.175 102.76 107.04TIP-39 PTHR2 0.0003934505 1.1032 −3.7746 100.33 100.71 VIP VIPR10.0003567715 1.9827 −0.96244 105 104.01 Compound 2 CALCR >0.1 0.070838Compound 2 CRHR1 >0.1 1.5625 Compound 2 CRHR2 >0.1 0.928 Compound 2PTHR1 >0.1 0.86197 Compound 2 PThR2 >0.1 2.2789 Compound 2 VIPR1 >0.10.60373

TABLE 12 Potency of compound 2 in human GIP receptor assay assay 1 assay2 EC₅₀ (nM) R square EC₅₀ (nM) R square GIP 2.31 1.00 1.28 0.99 Compound2 ≥100 ≥100 ≥100 ≥100 Oxyntomodulin ≥100 ≥100 ≥100 ≥100

Example 10: In Vivo Studies: Single-Dose Efficacy in DIO Mice

Food intake, body weight, glucose tolerance and plasma FGF21 levels wereassessed in DIO mice after a single dose of compound 2, or the GLP1Ragonist, dulaglutide. One day prior to dosing, animals were weighed andgrouped by BW. Mice were dosed as indicated by subcutaneous injection.The following morning, food was removed and the 6 hour fast was begun.BW and food weight (FW) were recorded. Fasting blood glucose wasmeasured and blood was collected for insulin measurement at 12:00 pm.One hour later, mice were dosed with glucose (1 g/kg, 20% glucose, 5mL/kg) intraperitoneally. An additional 20 μL of blood was collected viatail bleeding at 10 minutes for plasma insulin. Blood glucose levelswere measured by glucometer at 10, 30, 60, and 90 minutes after glucosechallenge. Mice were then euthanized with CO₂ and terminal blood sampleswere collected via cardiac puncture. The data are shown in Tables 13-19.

TABLE 13 Effect of compound 2 and dulaglutide on food intake (grams) inDIO mice over 18 hours after treatment Treatment 24 hours pre-dose 18hours post-dose Vehicle 3.1 ± 0.1 2.9 ± 0.1  Dulaglutide (0.3 nmol/kg)3.1 ± 0.1 1.5 ± 0.1*{circumflex over ( )} Compound 2 (1.0 nmol/kg) 3.2 ±0.1 2.5 ± 0.1{circumflex over ( )}# Compound 2 (2.0 nmol/kg) 3.4 ± 0.1 2.2 ± 0.1*{circumflex over ( )}# Compound 2 (4.0 nmol/kg) 3.2 ± 0.1 1.7± 0.1*{circumflex over ( )} Compound 2 (8.0 nmol/kg) 3.4 ± 0.1 1.4 ±0.2*{circumflex over ( )} *p < 0.01, versus Vehicle (18 hourspost-dose); {circumflex over ( )}p < 0.001, versus respective 24 hourspre-dose; #p < 0.0001, versus dulaglutide (0.3 nmol/kg) 18 hourspost-dose Two-Way ANOVA, Sidak's multiple comparisons test Valuesrepresent mean ± SEM for data from 8 animals per time point per group,except for Vehicle 24 hours pre-dose (n = 7)

TABLE 14 Effect of compound 2 and dulaglutide on percent body weightchange in DIO mice 18 hours after treatment Treatment Weight Change (%)Vehicle −0.7 ± 0.3  Dulaglutide (0.3 nmol/kg) −4.1 ± 0.3* Compound 2(1.0 nmol/kg) −1.9 ± 0.3*{circumflex over ( )} Compound 2 (2.0 nmol/kg)−2.8 ± 0.4*{circumflex over ( )} Compound 2 (4.0 nmol/kg) −4.4 ± 0.3*Compound 2 (8.0 nmol/kg) −5.2 ± 0.3*{circumflex over ( )} *p < 0.01,versus Vehicle; {circumflex over ( )}p < 0.05, versus dulaglutide (0.3nmol/kg) Two-Way ANOVA RM, Tukey's multiple comparisons test. Data onlyshown for 18 hour time point Percent weight change is calculatedrelative to body weights on day 0 (before dosing) Values represent mean± SEM for data from 8 animals per group

TABLE 15 Effect of compound 2 and dulaglutide on blood glucose (mg/dL)in DIO mice during an IPGTT 24 hours after treatment Time (min)Treatment 0 10 30 60 90 Vehicle 215 ± 9 353 ± 20 436 ± 19 424 ± 25 391 ±25 Dulaglutide 140 ± 7 264 ± 18 267 ± 22 228 ± 12 193 ± 9  (0.3 nmol/kg) Compound 2 169 ± 6 349 ± 17 411 ± 30 411 ± 19 384 ± 18 (1.0 nmol/kg) Compound 2  138 ± 11 288 ± 19 349 ± 27 303 ± 29 229 ± 20 (2.0 nmol/kg) Compound 2 114 ± 8 235 ± 23 237 ± 34 239 ± 30 195 ± 25 (4.0 nmol/kg) Compound 2  96 ± 7 206 ± 13 168 ± 22 166 ± 19 131 ± 12 (8.0 nmol/kg) Values represent mean ± SEM for data from 8 animals per time pointper group

TABLE 16 Blood glucose net AUC during an IPGTT in DIO mice 24 hoursafter treatment Treatment Blood Glucose Net AUC Vehicle 16539 ± 1400dulaglutide (0.3 nmol/kg)  8471 ± 1204* Compound 2 (1.0 nmol/kg) 19214 ±1596{circumflex over ( )} Compound 2 (2.0 nmol/kg) 13805 ± 1840 Compound2 (4.0 nmol/kg)  9827 ± 1046* Compound 2 (8.0 nmol/kg)  6035 ± 742* *p <0.05, versus Vehicle; {circumflex over ( )}p < 0.05, versus dulaglutide(0.3 nmol/kg) and JNJ-64151789 (4.0 and 8.0 nmol/kg) One-Way ANOVA,Tukey's multiple comparisons test Values represent mean ± SEM for datafrom 8 animals per group

TABLE 17 Effect of compound 2 and dulaglutide on 6 hour fasting glucose(mg/dL) in DIO mice, 24 hours after treatment Treatment Blood Glucose(mg/dL) Vehicle 215 ± 9  Dulaglutide (0.3 nmol/kg) 140 ± 7* Compound 2(1.0 nmol/kg) 169 ± 6* Compound 2 (2.0 nmol/kg)  138 ± 11* Compound 2(4.0 nmol/kg) 114 ± 8* Compound 2 (8.0 nmol/kg)  96 ± 7*{circumflex over( )} *p < 0.05, versus Vehicle; {circumflex over ( )}p < 0.05, versusdulaglutide (0.3 nmol/kg) One-Way ANOVA, Tukey's multiple comparisonstest Values represent mean ± SEM for data from 8 animals per group

TABLE 18 Effect of compound 2 and dulaglutide on plasma insulin levels(ng/ml) in DIO mice pre-glucose challenge (24 hours after treatment) and10 minutes post-glucose challenge during an IPGTT Plasma Insulin (ng/mL)Treatment Pre-glucose 10 minutes post-glucose Vehicle 2.7 ± 0.4 2.0 ±0.3 Dulaglutide (0.3 nmol/kg) 1.6 ± 0.2 1.7 ± 0.2 Compound 2 (1.0nmol/kg) 2.8 ± 0.4 1.8 ± 0.2 Compound 2 (2.0 nmol/kg) 2.3 ± 0.5 2.9 ±0.4 Compound 2 (4.0 nmol/kg) 2.1 ± 0.2 2.5 ± 0.5 Compound 2 (8.0nmol/kg) 1.5 ± 0.4 2.7 ± 0.3 Values represent mean ± SEM for data from 8animals per group

TABLE 19 Effect of compound 2 and dulaglutide on plasma FGF21 (ng/mL) inDIO mice 26 hours after treatment Treatment Plasma FGF21 (ng/mL) Vehicle0.4 ± 0.1 Dulaglutide (0.3 nmol/kg) 0.8 ± 0.2 Compound 2 (1.0 nmol/kg)0.7 ± 0.1 Compound 2 (2.0 nmol/kg) 1.5 ± 0.3 Compound 2 (4.0 nmol/kg)2.1 ± 0.4{circumflex over ( )} Compound 2 (8.0 nmol/kg)  3.5 ± 0.3* *p <0.0001, versus all groups; {circumflex over ( )}p < 0.05, versus allgroups except compound 2 (2.0 nmol/kg) One-Way ANOVA, Tukey's multiplecomparisons test Values represent mean ± SEM for data from 8 animals pergroup

Example 11: In Vivo Studies: Repeat Dosing in DIO Mice

The effects of a GLP1R/GCGR dual agonist, compound 2, and the GLP1Ragonist, dulaglutide in DIO mice were monitored during repeated dosingover 9 days. To control for the dosing frequency, all animals were doseddaily. Dosing frequency was determined on the basis of DIO mouse PK datafor compound 2 and dulaglutide. Animals in the dulaglutide groupreceived this drug daily. The vehicle-treated animals received vehicledaily. Those animals given compound 2 at 1.0, 2.0 or 4.0 nmol/kg weredosed with compound every third day and received vehicle on those dayswhen they were not injected with compound. The day prior to initiatingof dosing, animals were weighed and grouped by body weight. All micewere dosed as indicated by subcutaneous injection between 1-3 PM. Foodintake and body weight were monitored daily between 1-3 PM in thesetreatment groups.

Beginning 24-hr after the first dosing, three groups of mice werepair-fed (PF) to the 1.0, 2.0, or 4.0 nmol/kg compound 2-treatedanimals. The amount of food in the hopper of pair-fed mice was adjustedto match the average food consumed in the previous 24 hours by mice inthe compound treatment group to which they were being matched. All PFgroups were dosed daily with vehicle. On the ninth day (or Day 10 forthe pair-fed groups), animals were fasted for 6 hours and body weightand fasting glucose were measured. Blood was collected for measurementsof insulin, FGF21 and plasma lipids. The food intake, body weight, bodycomposition, fasting glucose, fasting insulin, fasting FGF21, andfasting plasma lipid data are shown in tables 20-26.

TABLE 20 Effect of compound 2 and dulaglutide on daily food intake(grams) in DIO mice over 9 days of treatment Time (days) Treatment 0 1 23 4 5 6 7 8 9 Vehicle 2.7 ± 0.1 2.9 ± 0.0 3.1 ± 0.1 2.9 ± 0.1 2.9 ± 0.13.0 ± 0.1 2.9 ± 0.1 3.0 ± 0.1 3.0 ± 0.1 2.9 ± 0.1 Dulaglutide 3.1 ± 0.21.5 ± 0.2* 1.3 ± 0.2* 1.7 ± 0.3* 1.9 ± 0.2* 2.1 ± 0.2* 2.2 ± 0.2* 2.3 ±0.2 2.9 ± 0.4 2.5 ± 0.2 (0.3 nmol/kg) Compound 2 3.1 ± 0.1 2.3 ± 0.1*#2.5 ± 0.1*# 2.6 ± 0.2 2.4 ± 0.1# 2.7 ± 0.2# 2.6 ± 0.1# 2.9 ± 0.2# 2.8 ±0.1# 2.7 ± 0.1 (1.0 nmol/kg) Compound 2 3.1 ± 0.1 2.0 ± 0.1* 2.0 ±0.1*{circumflex over ( )} 2.7 ± 0.1{circumflex over ( )} 2.5 ±0.2{circumflex over ( )} 2.3 ± 0.1 2.5 ± 0.2 2.7 ± 0.1 2.8 ± 0.3 2.7 ±0.1 (2.0 nmol/kg) Compound 2 2.9 ± 0.2 1.5 ± 0.1* 1.5 ± 0.2* 2.1 ± 0.2*1.7 ± 0.1* 1.6 ± 0.1* 1.6 ± 0.1* 1.8 ± 0.2* 2.2 ± 0.3*{circumflex over( )} 2.1 ± 0.1* (4.0 nmol/kg) *p < 0.05, versus Vehicle; {circumflexover ( )}p < 0.05, versus dulaglutide (0.3 nmol/kg); #p < 0.05, versuscompound 2 (4.0 nmol/kg) Two-Way ANOVA RM, Tukey's multiple comparisonstest All PF groups received average food intake amounts (data not shown)from respective treated groups. Values represent mean ± SEM for datafrom 6-10 animals per time point per group

TABLE 21 Effect of compound 2 and dulaglutide on percent body weightchange in DIO mice over 9 days of treatment Time (days) T 0 1 2 3 4 5 67 8 9 V 0.0 −0.1 ± 0.2 −0.1 ± 0.2* −0.2 ± 0.2*  −0.7 ± 0.3*  −0.5 ± 0.3* 0.2 ± 0.5*  0.1 ± 0.6*  0.0 ± 0.5*  −1.7 ± 0.6* 1 0.0 −3.7 ± 0.3 −6.2 ±0.6 −7.8 ± 0.8  −9.2 ± 0.8 −10.1 ± 0.8 −10.2 ± 0.8 −10.7 ± 1.0 −11.1 ±0.9 −13.1 ± 1.1 2 0.0 −1.9 ± 0.4 −2.9 ± 0.5{circumflex over ( )} −3.2 ±0.4{circumflex over ( )}  −4.4 ± 0.7 &  −4.7 ± 0.8 &  −4.5 ± 0.8 &  −5.3± 1.0 &  −5.2 ± 1.1 &  −6.6 ± 1.1 & 3 0.0 −1.6 ± 0.3 −2.7 ±0.4{circumflex over ( )} −3.5 ± 0.4{circumflex over ( )}  −3.8 ±0.3{circumflex over ( )}  −3.8 ± 0.4{circumflex over ( )}  −3.9 ±0.5{circumflex over ( )}  −3.5 ± 0.5{circumflex over ( )}  −3.4 ±0.5{circumflex over ( )}  −4.5 ± 0.5{circumflex over ( )} 4 0.0 −2.8 ±0.4 −4.7 ± 0.5 −4.9 ± 0.6{circumflex over ( )}  −7.1 ± 0.7  −8.0 ± 0.8 −8.0 ± 0.8  −9.1 ± 0.8# −10.0 ± 0.9# −11.5 ± 0.9# 5 0.0 −3.0 ± 0.4 −4.5± 0.4 −5.1 ± 0.5{circumflex over ( )}  −5.3 ± 0.5{circumflex over ( )} −5.8 ± 0.5{circumflex over ( )}  −6.1 ± 0.5{circumflex over ( )}  −5.8± 0.5{circumflex over ( )}  −6.2 ± 0.4{circumflex over ( )}  −6.9 ±0.4{circumflex over ( )} 6 0.0 −3.7 ± 0.3 −6.8 ± 0.3 −8.8 ± 0.5$ −11.6 ±0.6#$ −14.2 ± 0.7{circumflex over ( )}#$ −15.6 ± 0.9{circumflex over( )}#$ −18.4 ± 1.2{circumflex over ( )}#$ −20.4 ± 1.5{circumflex over( )}#$ −21.9 ± 1.6{circumflex over ( )}#$ 7 0.0 −3.6 ± 0.1 −5.6 ± 0.1−6.9 ± 0.1  −7.9 ± 0.2  −9.1 ± 0.2 −10.5 ± 0.3 −11.4 ± 0.3 −12.3 ± 0.3−13.3 ± 0.3 T: treatment; V: vehicle; 1: dulaglutide (0.3 nmol/kg); 2-3:compound 2 (1.0 nmol/kg); 4-5: compound 2 (2.0 nmol/kg); 6-7: compound 2(4.0 nmol/kg) *p < 0.01, versus all other treatments; {circumflex over( )}p < 0.05, versus dulaglutide (0.3 nmol/kg); #p < 0.05, versusrespective PF group; $p < 0.05, versus JNJ-64151789 (1.0 and 2.0nmol/kg); &p < 0.05, versus JNJ-64151789 (2.0 nmol/kg) Two-Way ANOVA RM,Tukey's multiple comparisons test. % Weight change is calculatedrelative to body weights on day 0 (before dosing) Values represent mean± SEM for data from 10 animals per time point per group

TABLE 22 Effect of compound 2 and dulaglutide on body composition (massand percent body weight) in DIO mice after repeated dosing Time (days)Mass (g) % of Body Weight Treatment Lean Fat Lean Fat Vehicle 24.0 ± 0.210.6 ± 0.5  57.5 ± 0.8 25.4 ± 0.8 Dulaglutide  22.1 ± 0.3* 7.6 ± 1.161.0 ± 1.7 22.8 ± 1.2 (0.3 nmol/kg) Compound 2 23.1 ± 0.5 9.5 ± 1.3 57.4± 2.5 23.1 ± 2.5 (1.0 nmol/kg) Compound 2 23.9 ± 0.3 10.6 ± 0.3  57.7 ±0.7 25.5 ± 0.5 (1.0 nmol/kg) PF Compound 2  22.2 ± 0.4* 8.2 ± 0.7 59.7 ±1.1 22.0 ± 1.6 (2.0 nmol/kg) Compound 2 23.4 ± 0.1 9.8 ± 0.4 58.5 ± 0.824.4 ± 0.8 (2.0 nmol/kg) PF Compound 2  20.9 ± 0.3* 7.4 ± 0.7 61.9 ± 2.421.6 ± 1.1 (4.0 nmol/kg) Compound 2 21.4 ± 0.6 9.3 ± 0.5 56.8 ± 1.4 24.7± 1.0 (4.0 nmol/kg) PF *p < 0.05, versus Vehicle One-Way ANOVA, Tukey'smultiple comparisons test Values represent mean ± SEM for data from 5animals per group

TABLE 23 Effect of compound 2 and dulaglutide on 6 hour fasting glucose(mg/dL) in DIO mice, after repeated dosing Treatment Blood Glucose(mg/dL) Vehicle 170 ± 5 Dulaglutide (0.3 nmol/kg)  123 ± 5* Compound 2(1.0 nmol/kg) 164 ± 5{circumflex over ( )} Compound 2 (1.0 nmol/kg) PF168 ± 7{circumflex over ( )} Compound 2 (2.0 nmol/kg)  140 ± 7* Compound2 (2.0 nmol/kg) PF 155 ± 6{circumflex over ( )} Compound 2 (4.0 nmol/kg)  98 ± 4*# Compound 2 (4.0 nmol/kg) PF  133 ± 6* *p < 0.05, versusVehicle; {circumflex over ( )}p < 0.05, versus dulaglutide (0.3nmol/kg); #p < 0.05, versus JNJ-64151789 (4.0 nmol/kg) PF One-Way ANOVA,Tukey's multiple comparisons test Values represent mean ± SEM for datafrom 10 animals per group

TABLE 24 Effect of compound 2 and dulaglutide on 6 hour fasting plasmainsulin (ng/mL) in DIO mice after repeated dosing Treatment PlasmaInsulin (ng/mL) Vehicle 3.0 ± 0.4  Dulaglutide (0.3 nmol/kg) 1.4 ± 0.2*Compound 2 (1.0 nmol/kg) 2.0 ± 0.3  Compound 2 (1.0 nmol/kg) PF 2.0 ±0.1* Compound 2 (2.0 nmol/kg) 1.1 ± 0.1*{circumflex over ( )} Compound 2(2.0 nmol/kg) PF 2.3 ± 0.3  Compound 2 (4.0 nmol/kg) 0.5 ± 0.1* Compound2 (4.0 nmol/kg) PF 1.2 ± 0.1* *p < 0.05, versus Vehicle; {circumflexover ( )}p < 0.05, versus compound 2 (2.0 nmol/kg) PF One-Way ANOVA,Tukey's multiple comparisons test Values represent mean ± SEM for datafrom 10 animals per group

TABLE 25 Effect of compound 2 and dulaglutide on 6 hour fasting plasmaFGF21 (ng/mL) in DIO mice after repeated dosing Treatment FGF21 (ng/mL)Vehicle 0.7 ± 0.1 Dulaglutide (0.3 nmol/kg) 0.5 ± 0.1 Compound 2 (1.0nmol/kg) 0.7 ± 0.2 Compound 2 (1.0 nmol/kg) PF 0.4 ± 0.1 Compound 2 (2.0nmol/kg) 0.6 ± 0.1 Compound 2 (2.0 nmol/kg) PF 0.2 ± 0.1 Compound 2 (4.0nmol/kg)  1.5 ± 0.3*{circumflex over ( )} Compound 2 (4.0 nmol/kg) PF0.3 ± 0.1 *p < 0.05, versus all groups; {circumflex over ( )}p < 0.05,versus compound 2 (4.0 nmol/kg) PF One-Way ANOVA, Tukey's multiplecomparisons test Values represent mean ± SEM for data from 10 animalsper group, except compound 2 (1.0 nmol/kg) and compound 2 (4.0 nmol/kg)PF where N = 9

TABLE 26 Effect of compound 2 and dulaglutide on plasma lipids in DIOmice after repeated dosing Time (days) Free Fatty Free Tri- Total AcidsGlycerol glycerides cholesterol Treatment (μmol/L) (mmol/L) (mg/dL)(mg/dL) Vehicle 66.2 ± 3.9 0.6 ± 0.0  30.1 ± 2.8 142.1 ± 5.2 Dulaglutide 73.2 ± 12.0 0.5 ± 0.1  24.1 ± 2.3 146.1 ± 3.3 (0.3 nmol/kg) Compound 271.8 ± 5.4 0.5 ± 0.0* 32.5 ± 4.5 149.1 ± 2.3 (1.0 nmol/kg) Compound 258.8 ± 6.1 0.6 ± 0.1  33.7 ± 4.1 161.4 ± 2.0 (1.0 nmol/kg) PF Compound 273.8 ± 4.6 0.5 ± 0.0* 30.4 ± 3.4  128.0 ± 6.6# (2.0 nmol/kg) Compound 2 84.8 ± 25.3 0.6 ± 0.0* 34.5 ± 3.3 149.9 ± 4.4 (2.0 nmol/kg) PF Compound2 71.2 ± 6.4 0.4 ± 0.0*  16.1 ± 2.0$  85.7 ± 4.9{circumflex over ( )}(4.0 nmol/kg) Compound 2 102.6 ± 14.5 0.7 ± 0.0  31.8 ± 1.1 137.8 ± 5.7(4.0 nmol/kg) PF *p < 0.05, versus compound 2 (4.0 nmol/kg) PF;{circumflex over ( )}p < 0.05, versus all other groups; #p < 0.05,versus compound 2 (1.0 nmol/kg) and compound 2 (2.0 nmol/kg) PF; $p <0.05, versus all groups except dulaglutide (0.3 nmol//kg) One-Way ANOVA,Tukey's multiple comparisons test Values represent mean ± SEM for datafrom 10 animals per group for free fatty acids, 8-10 animals per groupfor free glycerol, 9-10 animals per group for triglycerides and totalcholesterol

Example 12: In Vivo Studies: Single Dose Efficacy in Cynomolgus Monkeys

Biologics naïve cynomolgus monkeys were monitored daily for food intakeduring the three weeks prior to treatment. At the initiation oftreatment, animals were placed in four groups to achieve similar meanbody weight (n=8-9, mean body weight 7.4-7.9 kg, for each group).Animals in each group received a single subcutaneous dose correspondingto 1, 3, 5, or 7.5 nmol/kg of compound 2. Food consumption was monitoreddaily for an additional 14 days. Body weights were measured on the dayof dosing and 4, 7, 10, 14 and 21 days after dosing. The percentagechange in food intake was determined daily by comparing the food intakeon that day to the average daily food intake during the seven days priorto dosing. The results are provided in FIGS. 14 and 15.

Example 13: Pharmacokinetics (PK), Pharmacokinetic (PK)/Pharmacodynamics(PD) Analysis

DIO Mouse PK

The time-course of compound 2 was assessed in DIO mice (Table 27).Animals (n=3 per time point) were dosed s.c. at 10 nmol/kg.

TABLE 27 Time course of plasma exposures (nM) of compound 2 measured bybioassay and immunoassay Plasma exposures (nM) Time (hours) BioassayImmunoassay 0 N/A N/A 7  6.9 ± 2.0 16.7 ± 6.7 24 32.6 ± 3.5 71.0 ± 8.948 55.5 ± 2.4 111.9 ± 16.4 72 45.3 ± 4.0 95.7 ± 9.7 96  42.3 ± 14.5 86.8± 6.8 120 26.5 ± 6.9  58.1 ± 14.2 Values represent mean ± SEM for datafrom 3animals per time point per group

Rat PK

The pharmacokinetic parameters of compound 2 were assessed inSprague-Dawley rats. For determination of pharmacokinetic parameters(Table 28), bioassay-determined exposures were used from samplescollected at 0, 24, 48, 72, 144, 216, 312, 408, and 504 hours afterdosing.

TABLE 28 Pharmacokinetic parameters of compound 2 in Sprague-Dawley ratsParameter Subcutaneous Intraveneus C_(max) (nM) 5.7 ± 2.1 N/A T_(max)(hours) 64.0 ± 13.9 N/A AUC_(0-∞) (nM*hours) 456 ± 154  865 ± 185t_(1/2) (hours) 38.5 ± 12.8 26.2 ± 6.2 Data are mean +/− standarddeviation, exposures bioassay

Cynomolgus Monkey PK

The pharmacokinetic parameters of compound 2 were assessed in cynomolgusmonkey. Animals were dosed i.v. or s.c. at 6.45 nmol/kg. Fordetermination of pharmacokinetic parameters (Table 29),bioassay-determined exposures were used from samples collected at 0, 1,6, 24, 48, 72, 120, 240, 336, 432, and 528 hours after dosing.

TABLE 29 Pharmacokinetic parameters of compound 2 in cynomolgus monkeysParameter Subcutaneous Intraveneus C_(max) (nM) 89.1 ± 8.7 N/A T_(max)(hours)  56.0 ± 13.9 N/A AUC_(0-∞) (nM*hours) 11,700 ± 1380  14,700 ±5260 t_(1/2) (hours) 51.9 ± 6.2  55.9 ± 41.7 Values represent mean ±standard deviation

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the present description.

All documents cited herein are incorporated by reference.

1. An isolated monoclonal antibody or antigen-binding fragment thereofcomprising a heavy chain complementarity determining region 1 (HCDR1), aHCDR2, a HCDR3, and a light chain complementarity determining region 1(LCDR1), a LCDR2, and a LCDR3, having the polypeptide sequences of SEQID NO: 16, 17, 18, 19, 20, and 21, respectively.
 2. The isolatedmonoclonal antibody or antigen-binding fragment thereof of claim 1,wherein the isolated monoclonal antibody comprises a heavy chainvariable domain (VH) having the polypeptide sequence of SEQ ID NO:12,and a light chain variable domain (VL) having the polypeptide sequenceof SEQ ID NO:14.
 3. The isolated monoclonal antibody of claim 1, furthercomprising a Fc portion.
 4. The isolated monoclonal antibody of claim 3,comprising a heavy chain (HC) having the polypeptide sequence of SEQ IDNO:13, and a light chain (LC) having the polypeptide sequence of SEQ IDNO:15.
 5. An isolated nucleic acid encoding the monoclonal antibody orantigen binding fragment thereof of claim
 1. 6. A vector comprising theisolated nucleic acid of claim
 5. 7. A host cell comprising the vectorof claim
 6. 8. A method of producing an isolated monoclonal antibody orantigen binding fragment thereof, the method comprising culturing thehost cell of claim 7 under conditions to produce the monoclonal antibodyor antigen binding fragment thereof, and recovering the antibody orantigen-binding fragment thereof from the cell or culture.
 9. Aconjugate comprising a monoclonal antibody or antigen-binding fragmentthereof of claim 1 and at least one pharmacologically active moietyconjugated thereto.
 10. The conjugate of claim 9, wherein thepharmacologically active moiety is a therapeutic peptide.
 11. Theconjugate of claim 10, wherein the therapeutic peptide is conjugated tothe monoclonal antibody or antigen-binding fragment thereof at thecysteine residue of SEQ ID NO:18.
 12. The conjugate of claim 10, whereinthe therapeutic peptide is conjugated to the monoclonal antibody orantigen-binding fragment thereof via a linker.
 13. The conjugate ofclaim 12, wherein the linker comprises a peptide linker, a hydrocarbonlinker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG)linker, a polysaccharide linker, a polyester linker, or a hybrid linkerconsisting of PEG and an embedded heterocycle.
 14. The conjugate ofclaim 10, wherein the therapeutic peptide is selected from the groupconsisting of oxyntomodulin, glucagon-like peptide 1 (GLP1), exendin(exenatide), amylin (pramlintide), alpha-melanocyte stimulating hormone(MSH), cocaine- and amphetamine-regulated transcript (CART),neuropeptide Y receptor Y1 (NPY1) antagonists, neuropeptide Y receptorY5 (NPY5) antagonists, neurotensin S, neuropeptide B, neuropeptide W,ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystokinin(CCK), orexin, melanin-concentrating hormone (MCH), oxytocin, andstresscopin.
 15. The conjugate of claim 14, wherein the therapeuticpeptide is oxyntomodulin comprising the polypeptide sequence of SEQ IDNO:24.
 16. A method of producing the conjugate of claim 10, comprisingreacting an electrophile, introduced onto a sidechain of the therapeuticpeptide, with the sulfhydryl group of the cysteine residue of SEQ IDNO:18 of the monoclonal antibody or antigen-binding fragment thereof,thereby creating a covalent linkage between the therapeutic peptide andthe monoclonal antibody or antigen-binding fragment thereof.
 17. Apharmaceutical composition comprising the conjugate of claim 9 and apharmaceutically acceptable carrier.
 18. A method of producing apharmaceutical composition comprising the conjugate of claim 9, themethod comprising combining the monoclonal antibody or antigen-bindingfragment thereof with a pharmaceutically acceptable carrier to obtainthe pharmaceutical composition.
 19. A method of increasing the half-lifeof a therapeutic peptide in a subject, the method comprising conjugatingthe therapeutic peptide with a monoclonal antibody or antigen-bindingfragment thereof comprising a heavy chain complementarity determiningregion 1 (HCDR1), a HCDR2, a HCDR3, and a light chain complementaritydetermining region 1 (LCDR1), a LCDR2, and a LCDR3, having thepolypeptide sequences of SEQ ID NO: 16, 17, 18, 19, 20, and 21,respectively, wherein the therapeutic peptide is conjugated to themonoclonal antibody or antigen-binding fragment thereof at the Cysresidue of SEQ ID NO:18.
 20. The method of claim 19, wherein thetherapeutic peptide is selected from the group consisting ofoxyntomodulin, glucagon-like peptide 1 (GLP1), exendin (exenatide),amylin (pramlintide), alpha-melanocyte stimulating hormone (MSH),cocaine- and amphetamine-regulated transcript (CART), neuropeptide Yreceptor Y1 (NPY1) antagonists, neuropeptide Y receptor Y5 (NPY5)antagonists, neurotensin S, neuropeptide B, neuropeptide W, ghrelin,bombesin-like receptor 3 (BRS3), galanin, cholecystokinin (CCK), orexin,melanin-concentrating hormone (MCH), oxytocin, and stresscopin.
 21. Themethod of claim 16, wherein the electrophile is bromoacetamide ormaleimide.